HYPERGLYCOSYLATED hCG DETECTION DEVICE

ABSTRACT

The present invention related to a pregnancy test device that can selectively detect hyperglycosylated human chorionic gonadotropin (hCG-H) in a liquid sample. The sample can be deposited on a proximal portion of the device for transport to a distal portion of the device. The device can include a release medium formed of a first material and including a detectable label thereon and a capture medium, including a capture site, in fluid communication with the release medium and formed of a second, different material. At least one of the release medium and the capture medium includes a binding member that exhibits a moderate to high affinity for hCG-H and is selectively or preferentially reactive with hCG-H.

FIELD OF THE INVENTION

The present invention is related to devices that can selectively detecthyperglycosylated human chorionic gonadotropin (hCG-H) for detectingpregnancy in a woman. Another aspect of the present invention relates toa method of evaluating the viability of a pregnancy.

BACKGROUND OF THE INVENTION

Human chorionic gonadotropin (hCG) is a glycoprotein produced by theplacenta soon after fertilization. This hormone has the crucial role ofmaintaining steroid production by the corpus luteum in early pregnancy,and ensures that pregnancy progresses unabated. Thus, the detection ofhCG has been utilized as a marker for pregnancy in women. Accordingly,measurement of hCG in blood or urine has been the basis of prior andcurrent pregnancy tests or devices.

hCG

More specifically, the hCG hormone is a member of the glycoproteinhormone family (GPH) that includes luteinizing hormone (LH), folliclestimulating hormone (FSH), and thyroid stimulating hormone (TSH). Allmembers of the GPH family are heterodimers that consist of an alpha andbeta subunit. The alpha subunit is conserved across the GPHs, while thebeta subunit is unique. However, there remains about 80% homology acrossthe beta subunits. In addition to conferring differentiation, the betasubunit also grants each heterodimer its unique biological activity andreceptor specificity (Pierce and Parsons, 1981; Stenman et al., 2006).

hCG has a molecular weight of around 37 kDa. About one-third of thismass is due to glycosylation consisting of 8 oligosaccharide chainscovalently bound to the alpha and beta subunits (Pierce and Parsons,1981). It is produced by the syncytiotrophoblast cells followingfertilization and its primary function is to maintain the production ofprogesterone by the corpus luteum in early pregnancy (Hoshina et al.,1985; Lustbader et al., 1998). During excretion, intact hCG can bepartially degraded into its component subunits allowing differentvariants of hCG to be detected in liquid samples, such as urine.

As is well known, the association of hCG and pregnancy was firstreported in 1927. During the 1960's, the advent of immunoassays allowedthe direct detection of hCG in pregnancy urine (Wide and Gemzell, 1960;Vaitukaitis et al., 1972). A variety of different hCG isoforms orfragments are present in both serum and urine. Intact hCG has beendiscovered to be the predominant form of hCG and have the mostbiological relevance throughout all stages of pregnancy despite thepresence of the other isoforms detected include free hCG alpha subunit,free hCG beta subunit, hCG beta core fragment, nicked hCG, andhyperglycosylated hCG (hCG-H) (O'Connor et al., 1994; McChesney et al.,2005).

hCG-H

Although intact hCG is the most prevalent form of the molecule foundthroughout pregnancy, other distinct isoforms have emerged withdifferent biological activities. One such form, hyperglycosylated hCG(hCG-H), differs from regular hCG in the amount of and/or location ofoligosaccharide chains present on the beta subunit of hCG. Thisdifferent glycosylation pattern is believed to alter the function of themolecule such that hCG-H provides a role in promoting implantation of adeveloping zygote.

Whereas hCG production is typically considered to be limited topregnancy, it has also been found to be produced in certain types ofinvasive cancer associated with gestational trophoblastic disease (Coleet al., 2006). These tumors (hytatidiform mole, choriocarcinoma, andplacental site trophoblastic tumors) are both aggressive and malignant,and a key marker for their presence is the production of hCG(particularly the beta subunit of hCG) in the absence of pregnancy (Coleet al., 2003). Research into hCG produced by choriocarcinoma tumor cellsidentified that there were significant weight differences in hCG derivedfrom choriocarcinoma when compared to hCG present in pregnancy (˜40 kDacompared to ˜37 kDa) (Hussa, 1977; Mizuochi et al., 1983; Amano et al.,1988). Further studies on the choriocarcinoma derived hCG elucidatedthat the weight differences were due to excess glycosylation leading tosignificantly larger oligosaccharides on the beta subunit (Elliott etal., 1997). The term hyperglycosylated hCG, or hCG-H, was coined forthis higher molecular weight choriocarcinoma derived hCG containinginvasive properties, with regular hCG reserved for the hCG with normaloligosaccharide chains found during pregnancy (Elliott et al., 1997;Cole et al., 1998).

Previously researchers developed an hCG-H monoclonal antibody byimmunizing mice with hCG-H produced by a single patient withchoriocarcinoma. This antibody, B152, specifically recognizedchoriocarcinoma derived hCG-H in both serum and urine. This allowedresearchers to more effectively screen and monitor the presence ofhCG-H. Subsequent studies utilizing the B152 antibody found that hCG-His not only produced by choriocarcinoma, but it is also found in veryearly pregnancy as well (O'Connor et al., 1998), and that hCG-H is thepredominant form of hCG produced by cytotrophoblast cells at the time oftrophoblast invasion irrespective if the invasion is associated withchoriocarcinoma or pregnancy (Kovalevskaya et al., 2002).Cytotrophoblast cells differ from those that produce regular hCG(syncytiotrophoblast cells), and they confer a different function tohCG-H that is closely linked with the implantation of the zygote intothe uterine lining after conception (Kovalevskaya et al., 2002). Studieshave also shown that hCG-H cannot replace the corpus luteum stimulatingactivity of regular hCG which suggests a distinct biological activityfor hCG-H separate from that of pregnancy promoting function of regularhCG (Cole et al., 1991).

Early pregnancy derived hCG-H has the same molecular weight aschoriocarcinoma derived hCG-H (Kovalesyskaya et al., 2002), and themajority of all hCG immunoreactivity in serum and urine samples fromearly pregnancy is due to the presence of hCG-H (O'Connor et al., 1998;Cole et al., 1999; Butler et al., 2002; Cole et al., 2003; Sutton-Rileyet al., 2006). Further, hCG-H accounts for >90% of all hCG at the timeof implantation (Cole et al., 2003). This proportion of hCG-H steadilydecreases as pregnancy progresses until it only accounts for about 2% ofall hCG by the 2^(nd) and 3^(rd) trimesters.

Early Pregnancy Loss

There is a correlation between low or absent levels of hCG-H in earlypregnancy, and early pregnancy loss. In light of research implicatingthat hCG-H plays a key role in trophoblast invasion, many have suggestedthat the high incidence of early pregnancy loss in the absence of hCG-His due to ineffective implantation of the zygote in the uterine liningSome studies have estimated that only 30% of all fertilized eggscontinue to term to result in a live birth (Zinaman et al., 1996; Slamaet al., 2002). Over the years a subset of these unsuccessful pregnanciesthat result from a failed implantation have been given names such as‘occult pregnancies’, ‘preclinical pregnancies’, ‘biochemicalpregnancies’, and ‘early pregnancy loss’ (Macklon et al., 2002). Theyall serve to describe a phenomenon whereby the conceptus is unable tosuccessfully implant and therefore pregnancy does not progress past thefirst few weeks after conception. The incidence of early pregnancy lossdue to failed implantation has been estimated to be about 30% of allconceptions, making it a significant occurrence in fertile individualsattempting to conceive (Wilcox et al., 1988; Macklon et al., 2002).

Fertilization of the egg typically occurs in the fallopian tubeapproximately 24-48 hours after ovulation. The fertilized egg (nowtermed a zygote) continually divides as it travels through the fallopiantube. However, its survival is not ensured until it enters the uterinecavity and implants into the uterine lining. Implantation typicallyoccurs from about 7 to about 10 days after fertilization and is theresult of several complex molecular interactions that allow thedeveloping blastocyst to embed in the lining and eventually establishcontact with nutrient enriched maternal blood (Carson et al., 2000;Enders, 2000; Norwitz et al., 2001). hCG-H has been identified as amarker whose presence (or absence) can serve to indicate if thissuccessful implantation in the uterine lining has occurred (Cole andKhanlian, 2007).

For instance, many studies have found that an unduly low proportion (orabsence) of hCG-H in early pregnancy is associated with early pregnancylosses prior to the 6^(th) week of gestation (O'Connor et al., 1998;Kovalevskaya et al., 2002; Sutton-Riley et al., 2006). Sasaki et al.(2007) recently found that in 62 successful conceptions, only those thathad an hCG-H proportion greater than 50% in the first week followingconception continued to term. As such, low proportions of hCG-H aroundthe time of implantation may destine pregnancy for failure due to anunsuccessful implantation. Such studies, taken together with theabundance of choriocarcinoma data associating hCG-H with trophoblastinvasion, suggest that hCG-H has an essential role in promoting theinvasive properties of the conceptus resulting in successfulimplantation (Lei et al., 1999; Cole et al., 2007). In the absence ofthe implantation promoting ability of hCG-H, the conceptus may notachieve successful implantation and ultimately result in an earlypregnancy loss (Kovalevskaya et al., 2007; Cole, 2007).

hCG-H and Pregnancy Testing

Traditional pregnancy tests in both the point of care (POC) and over thecounter (OTC) markets are developed to detect regular or total hCG.Although both regular hCG and hCG-H can be used to measure pregnancy,they are not equal in the results that are conveyed to the consumer(Cole et al., 2007). As up to 30% of all conceptions result in earlypregnancy loss (Wilcox et al., 1988), by assaying for regular hCG alone,these non-viable pregnancies are being detected and conveyed to theconsumer as a successful pregnancy. As such, regular hCG can beconsidered a poor discriminator of pregnancies that may ultimatelyresult in early pregnancy loss. The overwhelming majority of currentlyavailable POC and OTC pregnancy tests poorly detect hCG-H, with only ahandful of tests displaying equal sensitivity to both regular hCG andhCG-H (Butler et al., 2001; Cole et al., 2003; Cole et al., 2004).

Another shortcoming of traditional pregnancy tests is readily realizedupon consideration of women who are attempting to conceive throughfertility treatments. Such women are typically administered regular hCGto mimic an LH surge and promote ovulation from mature ovarianfollicles. This exogenous hCG is gradually cleared from their systemover a period of about 10 days (Stenman et al., 1997), but it precludesthese women from taking a traditional pregnancy test as it would resultin them achieving a ‘false positive’ due to lingering exogenous hCG intheir system. If these individuals were to use a pregnancy test specificfor hCG-H, the exogenous regular hCG in their system would likely haveno bearing on the results conveyed through the test.

Currently, there is no OTC device available that can specifically detecthCG-H. However, there is an automated chemiluminescent hCG-H assay basedon the B152 antibody (Nichols Advantage immunoassay) which has beencleared by the FDA for use in pregnancy related applications (Pandain etal., 2003; Weinans et al., 2005). While it is specific for hCG-H, thistest requires a long sample incubation time of about 4 hours. Thus, thistest must be run in a laboratory setting in order to achieve results(Cole et al., 2004). As many doctors advise women who achieve a positivepregnancy test result at home to wait for at least 6 weeks prior toscheduling a visit, the use of the laboratory based assay to detecthCG-H is not practical as those with unsuccessful implantation relatedto a low prevalence of hCG-H may have already suffered an earlypregnancy loss prior to the doctor's visit. The development of an athome hCG-H based pregnancy test would rapidly convey to the consumerthat not only are they pregnant, but that their odds for early pregnancyloss resulting from failed implantation are significantly reduced.

There remains a need for both POC and OTC pregnancy test devices thatcan selectively or preferentially detect hCG-H. There also remains aneed for a pregnancy test device exhibiting an improved level ofaccuracy for determining the viability of a pregnancy.

BRIEF SUMMARY OF THE INVENTION

The present invention satisfies at least some of the aforementionedneeds by providing pregnancy devices that can selectively orpreferentially detect hyperglycosylated human chorionic gonadotropin(hCG-H). Embodiments of the present invention include a device forselectively or preferentially detecting hyperglycosylated humanchorionic gonadotropin (hCG-H) in a liquid sample deposited on aproximal portion of the device for transport to a distal portion of thedevice. This particular device includes a release medium formed of afirst material having a detectable label thereon and a capture medium influid communication with the release medium and formed of a second,different material. The capture medium includes a capture site. At leastone of the release medium and the capture medium includes a bindingmember that exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with hCG-H. Devices according toembodiments of the present invention can provide confirmation of theviability of a pregnancy as only fertilized eggs with a high likelihoodof successful implantation (i.e. those with an appropriate level ofhCG-H) can be detected by the assay.

In other embodiments, the invention comprises a device for selectivelyor preferentially detecting hyperglycosylated human chorionicgonadotropin (hCG-H), in which the device includes a release mediumformed of a first material and having a detectable label thereon. Thedevice also includes a capture medium in fluid communication with therelease medium and formed of a second, different material. The capturemedium includes a capture site. Devices according to this particularembodiment include a scavenger component that is selectively orpreferentially reactive with regular hCG. The scavenger component can belocated between the location of sample deposit and the capture site.Further, at least one of the release medium and the capture medium caninclude a binding member that is reactive with hCG-H. In a preferredembodiment the binding member can be selectively or preferentiallyreactive with hCG-H and also exhibit a moderate to high affinity forhCG-H.

In certain embodiments, the device for selectively or preferentiallydetecting hCG-H in a liquid sample deposited on a proximal portion ofthe device for transport to a distal portion of the device includes arelease medium formed of a first material and having a detectable labeland a capture medium in fluid communication with the release medium andformed of a second, different material. The capture medium includes acapture site. Additionally, such devices include a mixture of bindingmembers. In these embodiments, the mixture of binding members includes afirst group of binding members that are selectively or preferentiallyreactive with an epitope of regular hCG and a second group of bindingmembers that exhibit a moderate to high affinity for hCG-H and areselectively or preferentially reactive with an epitope of hCG-H. In onesuch embodiment, the binding members are selectively or preferentiallyreactive with hCG-H account for greater than 50% of the total number ofbinding members present in the mixture.

In yet another embodiment, the present invention comprises a device forselectively or preferentially detecting hCG-H in a liquid sample, inwhich the device includes a release medium formed of a first materialand having a detectable label and a capture medium in fluidcommunication with the release medium. Devices according to suchembodiments further include at least one binding member that exhibits amoderate to high affinity for hCG-H and is selectively or preferentiallyreactive with an epitope of hCG-H and at least one binding member thatis reactive with an epitope of regular hCG. The capture medium ispreferably formed of a second, different material, and includes a firstcapture site that directly or indirectly binds hCG-H and a secondcapture site that directly or indirectly binds regular hCG.

In certain embodiments, the present invention comprises a device forselectively or preferentially detecting hCG-H in a liquid sample,wherein the device includes a common fluid path for receiving anddistributing the liquid sample. The device also includes at least onerelease medium in fluid communication with the common fluid path. Therelease medium can be formed of a first material and include adetectable label. Devices according to these embodiments can include afirst capture medium in fluid communication with the at least onerelease medium, wherein the capture medium is preferably formed of asecond material. The first capture medium includes a capture site thatdirectly or indirectly selectively or preferentially binds hCG-H. Also,devices according to such embodiments can also preferably include asecond capture medium in fluid communication with the at least onerelease medium and formed of the same material as the other capturemedium. The second capture medium can include a capture site thatdirectly or indirectly binds regular hCG.

In another aspect, the present invention provides a method of evaluatingthe viability of a pregnancy. Methods according to embodiments of thepresent invention include providing a test device for selectively orpreferentially detecting hCG-H, as described herein, and applying aliquid sample potentially including one or both of regular hCG and hCG-Hto the device. Such methods can also include detecting the presence orlack thereof of hCG-H in the liquid sample. The detected presence ofhCG-H indicates a viable pregnancy.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily drawn toscale, and wherein:

FIG. 1 illustrates the relative hCG-H discrimination between antibodiesin a traditional lateral flow format;

FIG. 2A illustrates the relative hCG-H discrimination between antibodiesin an avidin biotin lateral flow format for liquid samples having 25mIU/ml standards;

FIG. 2B illustrates the relative hCG-H discrimination between antibodiesin an avidin biotin lateral flow format for liquid samples having 12.5mIU/ml standards;

FIG. 2C illustrates the relative hCG-H discrimination between antibodiesin an avidin biotin lateral flow format for liquid samples having 6.25mIU/ml standards;

FIG. 3A depicts a top view of one embodiment of a test device accordingto the present invention;

FIG. 3B depicts a longitudinal side view of an embodiment of a testdevice according to the present invention;

FIG. 3C depicts a bottom view of an embodiment of a test deviceaccording to the present invention;

FIG. 3D depicts a tail end view of an embodiment of a test deviceaccording to the present invention

FIG. 3E depicts a top perspective view an embodiment of a test deviceaccording to the present invention;

FIG. 4A depicts a front, top, left side perspective view of a preferredembodiment of a test device according to the present invention;

FIG. 4B depicts the test device according to the embodiment from FIG. 4Awith the cap thereof removed;

FIG. 4C depicts a top plan view of the test device embodiment from FIG.4A

FIG. 4D depicts a bottom plan view of the test device embodiment fromFIG. 4A

FIG. 4E depicts a left side elevational view of the test deviceembodiment from FIG. 4A

FIG. 5 depicts a schematic top view of a biphasic substrate including asample collector and upstream absorbent according to one embodiment ofthe invention;

FIG. 6 depicts a schematic top view of a biphasic substrate according toone embodiment of the invention;

FIG. 7 depicts a schematic top view of a biphasic substrate according toanother embodiment of the invention;

FIG. 8 depicts a schematic side view of the embodiment of a test deviceaccording to the invention illustrated in FIGS. 4A-4C and FIG. 5;

FIG. 9 depicts one embodiment that directly detects hCG-H;

FIG. 10 depicts another embodiment that directly detects hCG-H;

FIG. 11 depicts an embodiment including a scavenging component, wherethe device indirectly detects hCG-H;

FIG. 12 depicts another embodiment including a scavenging component,where the device indirectly detects hCG-H;

FIG. 13A depicts a multi-line test device indicating that the user isnot pregnant and that the device is functioning properly;

FIG. 13B depicts a multi-line test device indicating that the user ispregnant, that the pregnancy is viable, and that the device isfunctioning properly;

FIG. 13C depicts a multi-line test device indicating that the user ispregnant, that the pregnancy is not viable, and that the device isfunctioning properly

FIG. 14 depicts a multi-strip test device;

FIG. 15 depicts a multi-strip test device including a common releasemedium;

FIG. 16 shows Biacore sensorgrams for SMA 4D8 against normallyglycosylated hCG;

FIG. 17 shows Biacore sensorgrams for SMA 4D8 against free β hCG;

FIG. 18 shows Biacore sensorgrams for SMA 4D8 against recombinant hCG;

FIG. 19 shows Biacore sensorgrams for SMA 4D8 against JEG-3 hCG-H;

FIG. 20 shows the SMA 4D8 ELISA antigen fingerprinting to illustrate thedifferential recognition of hCG antigens by SMA 4D8;

FIG. 21 shows Biacore sensorgrams for SMA 3E8 against normallyglycosylated hCG;

FIG. 22 shows Biacore sensorgrams for SMA 3E8 against free β hCG;

FIG. 23 shows Biacore sensorgrams for SMA 3E8 against recombinant hCG;

FIG. 24 shows Biacore sensorgrams for SMA 3E8 against JEG-3 hCG-H;

FIG. 25 shows the SMA 3E8 ELISA antigen fingerprinting to illustrate thedifferential recognition of hCG antigens by SMA 3E8;

FIG. 26 shows Biacore sensorgrams for B152 against normally glycosylatedhCG;

FIG. 27 shows Biacore sensorgrams for B152 against free β hCG;

FIG. 28 shows Biacore sensorgrams for B152 against recombinant hCG;

FIG. 29 shows Biacore sensorgrams for B152 against JEG-3 hCG-H;

FIG. 30 shows the respective color intensity of particular embodimentsaccording to the present invention as compared to production devices;

FIG. 31 shows a summary of results for tests using particularembodiments conducted on hCG free urine samples of differing non-surgeLH values;

FIG. 32 shows a summary of results for tests using particularembodiments conducted on hCG free urine samples of differingconcentrations of LH surge samples;

FIG. 33 shows a summary of results for tests conducted using particularembodiments on of peri- and post-menopausal urine samples with varyinglevels of pituitary hCG;

FIG. 34 shows a set of data obtained from tests conducted usingparticular embodiments on well characterized clinical urine samples fromearly pregnancy;

FIG. 35 shows an additional set of data obtained from tests conducted onwell characterized clinical urine samples from early pregnancy; and

FIG. 36 shows data from tests conducted using particular embodiments onfour cycles of women who suffered early pregnancy loss (EPL).

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, the invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. As used in the specification, and in the appended claims,the singular forms “a”, “an”, “the”, include plural referents unless thecontext clearly dictates otherwise.

In one aspect, the present invention provides a pregnancy test device,such as an OTC or POC pregnancy test. The device includes a proximalportion in fluid communication with a distal portion. A liquid sample(e.g., urine) can be directly or indirectly deposited on the proximalportion of the device for transport to the distal portion. In general,embodiments of the present invention include a first binding memberlocated on the proximal portion of the device and a second bindingmember deposited on the distal portion of the device. Preferably, atleast one of the binding members exhibits a moderate to high affinityfor hCG-H while also being selectively or preferentially reactive withan epitope of the hCG-H. Such devices also include a capture sitelocated on the distal portion of the device that directly or indirectlybinds the hCG-H. The presence of hCG-H in the liquid sample can bedetermined by visual inspection of the capture site, where the presenceof hCG-H is indicated by the presence of color development at thecapture site. Accordingly, embodiments of the present invention providea device that selectively or preferentially detects hCG-H.

As used herein, “antibody” refers to a polypeptide substantially encodedby an immunoglobulin gene or immunoglobulin genes, or fragments thereof,which specifically recognize and bind an antigen. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon, and mu constant region genes, as well as the immunoglobulinvariable region genes. Antibodies include fragments, such as Fab',F(ab)₂, Fabc, and Fv fragments. The term “antibody,” as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAmethodologies, and further includes “humanized” antibodies made by nowconventional techniques.

An antibody “specifically binds to”, “is selectively reactive with” or“is selectively immunoreactive with” a protein or epitope thereof whenthe antibody functions in a binding reaction with the protein withoutsignificantly binding with other proteins. In order for the antibody tobind to a protein, the protein should contact the antibody. Accordingly,contacting a sample suspected of containing an antigen of interest withan antibody to the antigen will permit the antibody to bind the antigen.The binding of the antibody to the protein permits determination of thepresence of the protein in a sample in the presence of a heterogeneouspopulation of proteins and other agents. Thus, under designatedimmunoassay conditions, the specified antibodies selectively bind to aparticular protein and do not significantly bind to other proteinspresent in the sample (e.g., greater than 90% or 95% differentialdiscrimination). Specific binding to a protein under such conditionsrequires an antibody that is selected for specificity or selectivity fora particular protein. Several methods for determining whether or not apeptide is immunoreactive with an antibody are known in the art. Incertain embodiments, an antibody “preferentially binds to”, “ispreferentially reactive with” or “is preferentially immunoreactive with”hCG-H if the antibody exhibits greater than 50% differentialdiscrimination of hyperglycosylated hCG over regular hCG. In certainembodiments, an antibody “preferentially binds to”, “is preferentiallyreactive with” or “is preferentially immunoreactive with” hCG-H if theantibody exhibits greater than 60%, 70%, 80, or 90% differentialdiscrimination of hyperglycosylated hCG over regular hCG. Similarly, anantibody “preferentially binds to”, “is preferentially reactive with” or“is preferentially immunoreactive with” regular hCG if the antibodyexhibits greater than 50% differential discrimination of regular hCGover hyperglycosylated hCG. In certain embodiments, an antibody“preferentially binds to”, “is preferentially reactive with” or “ispreferentially immunoreactive with” regular hCG if the antibody exhibitsgreater than 60%, 70%, 80, or 90% differential discrimination of regularhCG over hyperglycosylated hCG.

In some embodiments, the present invention specifically differentiatesbetween hyperglycosylated hCG and “regular” hCG. As used herein,“regular” hCG should be understood as encompassing any form of hCG thatis not hyperglycosylated. Thus, regular hCG may encompass naturallyoccurring hCG that is not hyperglycosylated and/or recombinant hCG thatis not hyperglycosylated. Regular hCG may further be delineated as beingnormally glycosylated (i.e., the type and degree of glycosylationnormally found in the human body) or being non-glycosylated.

As used herein a “moderate to high affinity” antibody or binding memberthereof comprises an antibody or binding member that can bind with aparticular antigen within a relatively short incubation time. Likewise,an antibody or binding member thereof “exhibits a moderate to highaffinity” for a particular antigen should be understood as requiring arelatively short sample incubation time. For instance, an antibody orbinding member exhibits “moderate to high affinity” at equilibrium(“KA”) greater than 1e⁹ (e.g., 1e⁹ to 1e⁹, 1e⁹ to 1e¹⁰, or 1e¹⁰ to 1e¹²)or preferably at least 1e¹⁰ (e.g., 1e¹⁰ to 1e¹²). In certainembodiments, KA values at equilibrium between about 1e⁹ to 1e¹⁰ can bedeemed as exhibiting a moderate affinity, while KA values at equilibriumof at least 1e¹⁰ can be deemed as exhibiting a high affinity. Atequilibrium, KA values of 1e⁸ or less can be deemed as exhibiting a lowaffinity. Such KA values can be experimentally determined using anysuitable method known in the art. Specifically, such KA values can bedetermined by analysis with a Biacore (GE Healthcare) instrumentaccording to the procedure set forth in the Examples section herein.

As used herein, a “capture antibody” should be understood as anantibody, such as a monoclonal or polyclonal antibody, attached to asubstrate directly or indirectly, such as a solid substrate. The captureantibody can include at least one binding member that specifically orpreferentially binds a particular, distinct epitope of an antigen, suchas hCG-H or regular hCG.

According to embodiments of the present invention, a pregnancy testdevice is provided including at least one moderate to high affinityantibody which selectively or preferentially recognizes and binds hCG-H.Preferably, such moderate to high affinity antibodies exhibit limited tominimal binding to regular hCG or other hCG related molecules that maybe present in a liquid sample. For instance, two sheep monoclonalantibodies (SMA's) have been developed which selectively orpreferentially recognize and bind hCG-H. Examples of antibodies that areselectively or preferentially reactive with hCG-H that specifically maybe used according to the present invention are SMA 3E8 and SMA 4D8,which are available from Church & Dwight, Inc. (Princeton, N.J.). Forinstance, FIG. 1 provides normalized data for high affinity antibodies3E8 and 4D8 compared to that of a known antibody (i.e., CCFO1) when usedin a traditional lateral flow format. The legend for FIG. 1 lists hCG-H(i.e., hyperglycosylated hCG), hCG (normally glycosylated hCG), andrecombinant hCG (i.e., poorly-glycosylated hCG). FIG. 1 illustrates thatboth 3E8 and 4D8 display desirable discrimination between hCG-H andrecombinant hCG when compared to the prior art antibody used in currentpregnancy tests. In particular, the prior art antibody exhibited about a30% hCG-H discrimination while 3E8 exhibited about a 70% hCG-Hdiscrimination and 4D8 exhibited about an 85% hCG-H discrimination.

As shown in FIGS. 2A-2C, three antibody pairs (i.e., 3 separate deviceseach having a unique pair of antibodies) were compared to two differentversions of devices marketed by Church and Dwight Co., Inc. under thebrand name FIRST RESPONSE® Early Result. The two production devices werethe First Response Early Result pregnancy test (hereinafter referencedas “FR1” and in the Figures) and Upgraded First Response Early Resultpregnancy test (hereinafter referenced as “FR2” and in the Figures). TheFR1 devices utilize a labeled binding antibody (i.e., large particlesize gold as the label) striped on the release medium and monomericstreptavidin immobilized on the capture medium. The FR2 devices aresimilar to the FR1 devices except the FR2 devices use polymerizedstreptavidin.

FIGS. 2A-2C provide normalized data for devices according to certainembodiments in an avidin-biotin lateral flow format of the presentinvention compared to that of production devices currently available.Moderate to high affinity antibodies 3E8 and 4D8 were biotinylated andconverted to biotin probe striping solutions (0.1 mg/ml). These stripingsolutions were then striped on the release medium of a biphasic teststrip with either 11D6-2B10 large gold probe (OD30) or CCFO1 large goldprobe (OD30). The capture mediums were striped with 1.5 mg/mlpolymerized streptavidin and GAM. Each of the devices was separatelytested with urine samples containing 25, 12.5, or 6.25 mIU/ml of 4different hCG standards: JEG-3 derived hCG-H (i.e., hyperglycosylatedhCG), regular hCG (i.e., normally glycosylated hCG), recombinant hCG(i.e., non-glycosylated hCG), and WHO hCG beta subunit.

FIG. 2A shows the normalized data for each device tested with 25 mIU/mlof the four hCG standards. Each of the devices including an antibodyhaving a moderate to high affinity for hCG-H exhibited a differentialdiscrimination of hyperglycosylated hCG and recombinant hCG of around80%. FIG. 2B shows similar results for each device having been testedwith 12.5 mIU/ml of each standard. FIG. 2C shows similar results foreach device having been tested with 6.25 mIU/ml of each standard. Eachof FIGS. 2B and 2C illustrate the desirably increased level indifferential discrimination achieved according to these particularembodiments of the present invention.

Further, the biomolecular interactions between antibodies SMA 4D8 andSMA 3E8 and various antigens were analyzed to evaluate the relativespecificity and affinity of each antibody for each antigen. Thus, an‘antigen fingerprinting’ has been performed for both antibodies; theresults of which are discussed in detail in the Examples section. Asdiscussed in the Examples section, antibodies that show a moderate tohigh affinity for hCG-H and a reduced or lower affinity for otherantigens, such as SMA 4D8 and SMA 3E8, are particularly useful accordingto the invention.

Embodiments of the present invention preferably make use of a conjugatecomprising an antibody bound to a detectable label component (which canbe colored particles, such as a metal sol or colloid, preferably gold orlatex beads or soluble dyes). The conjugate can take two distinct forms,depending on whether the assay is designed to exploit the “sandwich” or“competitive” technique.

Any detectable label recognized in the art as being useful in variousassays could be used in the present invention. In particular, thedetectable label component can include compositions detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. As such, the label component produces a detectable signal. Forinstance, suitable labels include soluble dyes, fluorescent dyes,chemiluminescent compounds, radioisotopes, electron-dense reagents,enzymes, colored particles, or dioxigenin. The label component cangenerate a measurable signal, such as radioactivity, fluorescent light,color, or enzyme activity, which can be used to identify and quantifythe amount of label bound to a capture site. Thus, the label componentcan also represent the presence or absence of a particular antigen boundthereto.

In certain embodiments, the label component preferably comprises a goldcolloid having a mean particle size of about 50 nm to about 100 nm priorto formation of the labeled conjugate More preferably, the mean particlesize can range from about 60 nm to about 80 nm prior to formation of thelabeled conjugate

In embodiments wherein the device of the invention makes use of asandwich technique, the antibody used in the detection comprises abinding member or site which binds to an epitope on the analyte fordetection, such as hCG-H. The antibody preferably has a label componentbound thereto to provide a labeled antibody. The labeled antibody reactswith the analyte to form a complex in the liquid sample. The analyte,which is bound with the labeled antibody, reacts with a capture antibodyto form a “sandwich” comprising the capture antibody, analyte, and thelabeled antibody. This sandwich complex is progressively produced as thetest liquid with the analyte therein continuously moves along the teststrip of the device. As more and more analyte/labeled antibody complexis immobilized as a sandwich with the capture antibody at the capturesite, the label components aggregate and become visible through aviewing window, indicating the presence of a particular analyte in theliquid sample.

Embodiments of the invention can include one or more standards orinternal controls that allow for determination of whether signaldevelopment (e.g., color development) is a true indication of thepresence or absence of analyte (e.g., hCG-H) in the sample or is simplyan artifact, such as caused by nonspecific sorption. For example, in oneembodiment employing the sandwich technique, the standard consists of anegative control site, preferably disposed adjacent the test site, andvisible through a second window proximate the first. The negativecontrol site preferably is prepared identically to the test site, exceptimmobilization of the binding protein is omitted. Therefore, althoughthe conjugate will reach the site, it aggregates due only tonon-specific binding. If the test site is not appreciably more intensein color than the negative control site, the assay is considerednegative.

In certain embodiment, the device can include a positive control. Thus,when exploiting the sandwich technique for example, a cell may have anauthentic sample of the analyte for detection immobilized at a controlsite. If no color develops at this control site, the assay is consideredinconclusive. When exploiting the competitive technique, the developmentof color at the positive control site means the assay results areinconclusive.

In yet another embodiment, which can be particularly useful when thepregnancy test device comprises a biphasic test strip medium, thebiphasic medium comprises a control site disposed on the capture mediumdownstream of the capture site. The control site has immobilized thereonat least one capture antibody (e.g., a protein). The primary function ofthe control site is to capture and immobilize labeled antibody which hasnot been captured at the capture site.

According to various embodiments, the control site can includepolyclonal antisera specific for the labeled antibody immobilizedthereon. Indication of the presence of the label component at thecontrol site indicates proper functioning of the test, irrespective ofthe presence or absence of analyte in the sample. Preferably, both thecapture and control sites are visible through the window of the casing.In a preferred embodiment, the inventive device incorporates a biphasicchromatographic medium (or test strip) which enhances the speed andsensitivity of the assay. Generally, a biphasic substrate element usefulaccording to the invention comprises a release medium joined to acapture medium located downstream of the release medium. The release andcapture media preferably comprise two different materials or phaseshaving different specific characteristics. The two phases are joinedtogether to form a single liquid path such that a solvent front cantravel unimpeded from the proximal (upstream) end of the release medium(which can be defined as a proximal portion of the biphasic medium) tothe distal (downstream) end of the capture medium (which can be definedas a distal portion of the biphasic medium).

Reagents for detecting, labeling, and capturing an analyte of interestare disposed on the release and capture media. In certain embodiments, alabeled conjugate is located on the release medium and includes abinding member reactive with a particular site (sometimes referred to asa “first epitope”) on the analyte of interest. The labeled conjugatefurther comprises a detectable marker (or label), preferably colloidalgold. A capturable conjugate can be located on the release mediumdownstream of the binding member, which conjugate comprises an antibodywith a binding agent reactive with another particular site (sometimesreferred to as a “second epitope”) on the analyte of interest. The firstepitope and the second epitope are preferably different sites on theanalyte. The capturable conjugate also comprises one member of anaffinity pair and is capable of forming a complex with the labeledbinding member and the analyte. The labeled conjugate and the capturableconjugate both are releasably bound to the release medium such that whenthe solvent front created by the liquid sample being analyzed passesthrough the release medium, the labeled conjugate and the capturableconjugate both become solubilized by the liquid and flow with thesolvent along the liquid path. In operation, if any analyte is presentin the liquid sample, it reacts first with the labeled conjugate, thenwith the capturable conjugate as the front advances along the liquidpath to form a diffusible sandwich complex which is then transported bycapillary action. Thus, by the time the solvent front reaches thecapture medium section of the biphasic material, the capturable sandwichcomplex has formed.

In embodiments such as those described above, the capture mediumcontains the reagents used to capture the complex described above.Generally, the reagents are located on a capture site and comprise theother member of the affinity pair specific for the moiety comprising thecapturable conjugate and a reagent specific for the labeled bindingmember. Upon diffusion into the capture medium, the diffusible sandwichcomplex becomes concentrated by the interaction of the capture affinitymember with the capturable affinity moiety yielding a visual signal. Theaffinity member is immobilized, preferably by simple adsorption, at thecapture site, and does not advance with the solvent front.

The release medium can be formed from a substance which allows forrelease of indicator reagents. In certain embodiments, the releasemedium comprises a bibulous, hydrophilic material, such as absorbentmaterials. Preferred materials for use as a release medium include, butare not limited to, cotton linter, cellulosic materials, or materialsmade of cellulose together with a polymeric fibrous material, such aspolyamide or rayon fibers, and glass fiber material. The primaryfunction of the release medium is first to support and to subsequentlyrelease and transport various immunological components of the assay,such as a labeled conjugate and/or a capturable conjugate, both of whichare capable of binding to the analyte of interest. This release andtransport occurs during routine operation of the assay. Generally, therelease medium can be formed of any material capable of performing thefunction of holding, releasing, and transporting various immunologicalparts of the test such as the labeled test component.

Specific, non-limiting examples of materials useful in forming therelease medium include: cotton linter paper, such as S&S 903, S&S GB002,and BFC 180 (available from Whatman, Fairfield, N.J.); cellulosicmaterials, such as Grade 939 made of cellulose with polyamide, Grade 989made of cellulose blend fiber, and Grade 1278 and Grade 1281 made ofcellulose and rayon with polyamide (available from Ahlstrom Corporation,Mt. Holly Springs, Pa.); and glass fiber, such as Lydall borosilicate(available from Lydall, Inc., Rochester, N.H.). The release mediumpreferably is coated with an aqueous solution containing bovine serumalbumin (BSA) and a nonionic surfactant, such as Triton X-100 (availablefrom Rohm & Haas Co., Philadelphia, Pa.) in order to prevent nonspecificbinding and facilitate release of the diffusible reagents. A combinationof about 3% BSA and about 0.1% Triton X-100 is useful for this purpose.

The capture medium can be formed from a substance which permitsimmobilization of reagents for detection of the presence of analyte inthe test fluid. The capture medium generally comprises hydrophilicpolymeric materials, such as microporous films or membranes, whichpermit protein reagents to be immobilized directly on the membrane bypassive adsorption without the need for chemical or physical fixation.Of course, the use of chemical or physical fixation is not precluded bythe invention, and any known method for immobilizing the reagents to themembrane can be used.

Non-limiting examples of materials useful as the capture medium comprisea microporous polymeric film of nitrocellulose, nylon (e.g., nylon 66),or similar materials, or combinations of such materials. Materials foruse as the capture medium preferably have a pore size in the range offrom about 5 μm to about 20 μM. In specific embodiments, thenitrocellulose membrane may be nitrocellulose alone or a mixed ester ofnitrocellulose, such as in combination with an ester of nitric acidand/or other acids. The nitrocellulose membrane preferably is coated orlaminated onto a translucent or transparent polymeric film to providephysical support for the membrane.

In a preferred embodiment, a nitrocellulose polymer which has been castonto a polyester film, such as MYLAR®, is used. Alternatively, anitrocellulose membrane laminated onto a polyester film also may beused, although other backing materials besides polyester may be used.Pre-laminated or pre-cast sheets useful in the present invention arecommercially available, for example, from Millipore Corporation,Bedford, Mass. and Sartorius Corporation, Edgewood, N.Y.

In one embodiment, the release medium and capture medium are joined byoverlapping the downstream edge of the release medium over the upstreamedge of the capture medium, then adhering the resulting biphasicmaterial to a clear polymer film or opaque sheet, thereby holding themedia in place. The overlapping region allows for the efficient andrapid transfer of analyte containing fluid from the release medium tothe capture medium.

While the rapid transfer associated with the overlapping region isuseful, the manufacturing issues associated with reproducibly generatinga small overlapping region, such as necessary with small devices, can bedifficult. Therefore, in certain embodiments, the invention alsoprovides a test device having a biphasic design as described herein butwherein the release medium and the capture medium do not overlap butrather are connected by a non-overlapping butt joint. In suchembodiments, the fluid front moving along the test strip is transferredfrom the release medium to the capture medium through bridging thenon-overlapping region by capillary action.

Beneficially, the butt joining of the phases can maintain the sameefficacy of the overlapping of the phases, even after accelerated agingof the devices. Thus, the use of a butt joint simplifies the manufactureof the present test device without any loss of performance in thedevice.

The diffusible and non-diffusible reagents can be applied to the releaseand capture media, respectively, by any suitable technique. In oneembodiment, the diffusible reagents are applied to the release medium bydirect application onto the surface of the medium and dried to form anarrow band.

In one preferred embodiment, the device comprises a casing defining asample inlet, a test volume, and reservoir volume. Disposed within thecasing are a sample absorbent, the biphasic chromatographicsubstrate(s), and reservoir absorbent. The sample absorbent ispreferentially disposed within the casing and extending to the exteriorthereof. Located downstream of the sample absorbent is the biphasicchromatographic substrate comprising a release medium and a capturemedium joined together to form a single liquid path. The release andcapture media can be laminated onto a transparent plastic film or sheet.

The sample absorbent preferably is a bibulous hydrophilic material whichfacilitates absorption and transport of a fluid sample to the biphasicchromatographic medium. Such materials may include cellulose acetate,hydrophilic polyester, and other materials having similar properties.Further, a combination of absorbent materials also may be used.Non-limiting examples of useful materials include bonded celluloseacetate, bonded polyolefin, or hydrophilic polyester, such as thosematerials commercially available from Filtrona Fibertec Company(Colonial Heights, Va.). Other useful materials include absorbentmatrices, such as Grade 939, Grade 989, Grade 1278, or Grade 1281,available from Ahlstrom Corporation. The sample absorbent preferably iscoated with a buffered solution containing BSA and a nonionicsurfactant, such as Triton X-100. The presence of BSA and surfactantminimize non-specific adsorption of the analyte. A concentration ofabout 1% BSA and about 0.2% surfactant in tris buffer can be effectivefor this purpose.

By providing a reservoir of sorbent material disposed beyond thechromatographic substrate, a relatively large volume of the test liquidand any analyte it contains can be drawn through the test area to aidsensitivity. The reservoir absorbent generally facilitates capillaryaction along the chromatographic substrate and absorbs excess liquidcontained within the device. The reservoir absorbent preferablycompromises absorbent paper made from cotton long linter fibers, such asCF3, CF4, CF5, or 470 (available from Whatman) or cellulosic materials,such as Grade 3mM (available from Whatman) and Grade 320 (available fromAlhstrom).

In using a device according to various embodiments of the invention, theproximal portion of the biphasic substrate is contacted with the liquidsample being analyzed, wherein the liquid sample can be collected eitherdirectly or through the absorbent material comprising the samplecollector. The casing of the device may be configured to permit directcontact with a body fluid or as a dipstick for dipping in a container ofbody fluid or other test solution. The liquid sample travels impelled bysurface effects such as by capillary action along the liquid path formedby the substrate. More specifically, the test sample passes through thebiphasic chromatographic substrate and into reactive contact with thetest site (and optionally one or more control sites). Preferably, atleast the test site is visible to a user, such as through one or morewindows in the device's exterior casing. In a preferred embodiment, thelabeled binding member recognizing the analyte is disposed in preservedform on the release medium in the flow path within the device.

In one embodiment, if the analyte of interest is present in the sample,it passes through the inlet and the interior of the device where itsequentially reacts with the labeled antibody and the capturableantibody with the affinity agent, thereby forming the capturablecomplex. The complex formed by the analyte, labeled antibody, and thecapturable antibody then reacts with the immobilized capture componentat the capture site, the capture component being specific for theaffinity agent on the capturable antibody. This process results in thelabeled complex accumulating at the capture site. The presence of theanalyte is determined by observing the presence of the detectable markerat the capture site. If no analyte is present in the sample, thecapturable complex does not form and no detectable marker will bepresent at the capture site. If a control site is present, the unboundcomplex or the free labeled binding member will accumulate at thecontrol site.

In yet another embodiment, if the analyte of interest is present in thesample, it passes through the inlet and the interior of the device whereit reacts with a labeled antibody which is releasably attached to therelease medium. The liquid sample wicks up the release medium and faunsa sandwich complex with a capture antibody which is immobilized on thecapture medium and defining a capture site. As the sample front passesacross the capture sites, a complex is formed comprising the analyte,labeled antibody, and the capture antibody. Preferably, at least one ofthe labeled antibody and the capture antibody includes a binding memberthat exhibits a moderate to high affinity for hCG-H. This processresults in the labeled complex accumulating at the capture site. Thepresence of the analyte (e.g. hCG-H) is determined by observing thepresence of the detectable marker at the capture site. If no analyte ispresent in the sample, the complex does not form and no detectablemarker will be present at the capture site. If a control site ispresent, the free labeled binding member will accumulate at the controlsite.

Illustrations of one embodiment of a test device 5 according to thepresent invention are shown in FIGS. 3A-E. The test device 5 comprisesan outer, molded casing 10 which defines a hollow, elongate enclosure.The casing 10 includes a test liquid inlet 14 and an opening 16comprising a window through which the capture site (and control site, ifapplicable) is visible. As illustrated in FIGS. 3A-E, the window 16 isdisposed on a side of the casing 10 opposite the sample inlet 14. Thisconfiguration reduces the incidence of contamination of the test sitewhich is disposed in the interior of casing 10 and is exposed throughthe window 16. The casing 10 further defines vent openings 38, 40, and42 located along the sides and at the distal portion of the casing 10.The vent opening 38 reduces the incidence of “vapor lock” within thedevice during use. The presence of the openings 40 and 42 help to reduce“flooding” of the chromatographic substrate, which may occur when theuser applies too much sample to the device.

A preferred embodiment of the test device 5 is illustrated in FIGS.4A-E. As seen therein, the test device 5 comprises an outer, moldedcasing 10 which defines a hollow, elongate enclosure. The casing 10includes an opening 16 comprising a window through which the capturesite (and control site, if applicable) is visible. The test device 5further includes a test liquid inlet 14, which is covered by a removablecap 60. In this embodiment, the test liquid inlet 14 is external to thecasing 10 and is covered by the cap 60 except when in use. Providing thetest liquid inlet 14 external to the casing 10 allows for ease ofapplication of the test liquid to the test device 5, such as by placingthe test liquid inlet 14 in the path of a urine stream or dipping in acontainer holding the test liquid. The cap 60 is re-attachable (such as“snap-fitting” onto the lip 62 extending from the casing 10) and can bereplaced after application of the test liquid to avoid contamination ofthe sample while the test is proceeding. The test liquid inlet 14external to the casing can be a portion of the absorbent material 12, asillustrated in FIG. 5 and described below. In further embodiments, thetest liquid inlet 14 can be a portion of the biphasic chromatographicsubstrate 18. The casing 10 further includes a test strip support 70located on the bottom surface of the casing 10.

A specific embodiment of the assay materials for use according to theinvention is illustrated in FIG. 5. When the device is fully assembled,the assay materials of FIG. 5 preferably are disposed inside a casing.The assay materials comprise an absorbent material 12, a biphasicchromatographic substrate 18, and a reservoir material 24. The assaymaterials and the interior of the casing together define a flow path.When the inlet 14 is disposed within or otherwise in contact with aliquid sample, the liquid is transported by capillary action, wicking,or simple wetting along the flow path downstream through the absorbent12, along the chromatographic substrate 18, and into the reservoir 24,generally as depicted by the arrow. The absorbent material also servesas a filter which can remove from impure test samples particulate matterand interfering factors.

Illustrated in FIG. 6 is a biphasic chromatographic substrate 18,comprising a release medium 30 and a capture medium 32. The horizontaldashed line represents the interface between the release medium 30 andthe capture medium 32. As previously noted, this interface can be in theform of an overlapping relationship. Alternatively, the release medium30 can be butted up to the capture medium 32. Releasably disposed on therelease medium 30 is a band 26 of labeled binding member, e.g., anantibody-metal sol. In one embodiment, the labeled biding member is indehydrated form. As the liquid sample moves past the band 26, thelabeled binding member becomes entrained in the liquid, reconstituted(in the case of a dehydrated binding member), and reacts or competeswith any analyte present in the liquid sample. Disposed downstream ofthe labeled binding member is a band 28 of preferably dehydratedcapturable complex. The capturable complex comprises a binding memberwhich binds to a second epitope of the analyte, e.g. an antibody, and acapturable affinity component, e.g. biotin. The capturable complex alsobecomes entrained in the liquid sample as it advances along thesubstrate 18.

Immobilized on the capture medium 32 are, respectively, the capture site34 and the control site 36. In FIG. 6, the control and capture sites areillustrated as being disposed serially along the flow path.Alternatively, the control and capture site or sites may be disposedside by side, perpendicular to each other, or in other spatialrelationships. The capture site 34 comprises a quantity of a captureaffinity member specific for the capturable affinity component disposedon the release medium. For example, when the capturable affinity memberis biotin, the capture component may be streptavidin. Of course, anysuch complementary system of components could be used in place of biotinand streptavidin. The control site 36 typically comprises immobilizedantisera, antibody, or a protein binder such as Protein A or Protein Gcapable of binding the labeled binding member.

In certain embodiments, as illustrated in FIG. 7, a band 26 of labeledbinding member, e.g., an antibody-metal sol., can be releasably disposedon the release medium 30. In one embodiment, the labeled binding memberis in dehydrated form. As the liquid sample moves past the band 26, thelabeled binding member becomes entrained in the liquid, reconstituted(in the case of a dehydrated binding member), and reacts or binds with aparticular analyte or analytes of interest present in the liquid sample.Accordingly, the resulting conjugate comprising a binding antibody, alabel component, and an analyte for identification (e.g., hCG-H)advances along with the sample front until the reaching the capture site34. In this particular embodiment, the capture site includes at leastone immobilized capture antibody having a binding member which binds toa second epitope of the analyte. Accordingly, a “sandwich” including thedesired analyte is formed at the capture site 34. If desired, a controlsite 36 can include immobilized antisera, antibody, or protein such asProtein A or Protein G capable of binding the labeled binding member.

A side view of one embodiment of the operative portion of the assaymaterials is schematically illustrated in FIG. 8. As shown, theabsorbent material 12 is disposed proximate the release medium 30, andoverlaps the release medium 30 at one end. The release medium 30 in turnoverlaps the capture medium 32, which is disposed distal to the releasemedium 30. Again, the release medium 30 and the capture medium 32 mayalternatively be connected via a butt joint rather than being inoverlapping connection. The reservoir 24 overlaps the distal portion ofthe capture medium 32. These four components together form a singlefluid path, and they cooperate to cause sample liquid to flow from theabsorbent 12 along the release medium 30 and the capture medium 32 intothe reservoir 24.

The invention is not limited by the precise nature of the capture site34 and the corresponding control site 36, and in fact, the control site36 may be entirely eliminated if desired. Generally, antibody or otheraffinity agent can be immobilized at the capture site 34 and the controlsite 36 using absorption, adsorption, or ionic or covalent coupling, inaccordance with methods known per se. The capture medium 32 preferablyis selected to bind the capture reagents without the need for chemicalcoupling. Nitrocellulose and nylon both permit non-chemical binding ofthe capture component and control reagent.

Disposed downstream of the capture medium 32 is the reservoir 24comprising a relatively large mass of absorbent or superabsorbentmaterial. The purpose of reservoir 24 is generally to ensure that areasonably large amount of test liquid is drawn across thechromatographic medium. In certain embodiments, the sample absorbent 12can be omitted, and the release medium 30 can itself act as the sampleabsorbent. Such embodiments of the assay materials are useful inperforming dipstick assays.

Direct Detection

In one aspect, the present invention provides devices wherein detectionincludes directly binding hCG-H. In such embodiments, at least one ofthe release medium and the capture medium includes a binding member thatexhibits a moderate to high affinity for hCG-H and is selectively orpreferentially reactive with hCG-H.

In various embodiments, the release medium includes a labeled conjugatecomprising the detectable label and a first binding member that isreactive with at least one epitope of hCG-H or an epitope of regularhCG. The capture medium includes a capture site comprising a secondbinding member that is reactive with at least one epitope of hCG-H or anepitope of regular hCG. However, at least one of the first bindingmember and the second binding member preferably exhibits a moderate tohigh affinity for hCG-H and is selectively or preferentially reactivewith hCG-H. For example, in one embodiment, only the first bindingmember exhibits a moderate to high affinity for hCG-H and is selectivelyor preferentially reactive with hCG-H while in another embodiment onlythe second binding member exhibits a moderate to high affinity for hCG-Hand is selectively or preferentially reactive with hCG-H. Alternatively,certain embodiments can include a first binding member and a secondbinding member that both exhibit a moderate to high affinity for hCG-Hand are selectively or preferentially reactive with hCG-H.

As illustrated by FIG. 9, the device for selectively detecting hCG-H ina liquid sample, according to one embodiment, comprises a biphasicsubstrate 18. The biphasic substrate 18 can include a release medium 30formed of a first material and comprising a region including a labeledantibody 26 that includes a binding member reactive with a first epitopeof hCG-H or regular hCG and a capture medium 32 in fluid communicationwith the release medium. Preferably, the capture medium 32 can be formedof a second, different material, and include a capture site 34 having animmobilized capture antibody thereon. The immobilized capture antibodycan comprise a member reactive with a second epitope of hCG-H or regularhCG, such that if hCG-H is present in the sample, a sandwich complex isformed comprising the labeled antibody, hCG-H, and the immobilizedcapture antibody. In one preferred embodiment, at least one of thelabeled antibody and the immobilized capture antibody includes a bindingmember that exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with hCG-H. According to one suchembodiment, the immobilized capture antibody comprises a binding memberthat exhibits a moderate to high affinity for hCG-H and is selectivelyor preferentially reactive with hCG-H. In another embodiment, thelabeled antibody comprises a binding member that exhibits a moderate tohigh affinity for hCG-H and is selectively or preferentially reactivewith hCG-H. In one alternative embodiment, the labeled antibody includesa binding member that exhibits a moderate to high affinity for hCG-H andis selectively or preferentially reactive with a first epitope of hCG-Hand the immobilized capture antibody also comprises a member thatexhibits a moderate to high affinity for hCG-H and is selectively orpreferentially reactive with a second epitope of hCG-H.

According to certain embodiments, at least one of the capture antibodyor the labeled antibody comprises SMA 3E8. In other embodiment, at leastone of the capture antibody or the labeled antibody comprises SMA 4D8.Alternatively, one embodiment of the invention comprises a releasemedium having releasably attached thereto a labeled antibody comprisingSMA 3E8 and a capture medium having immobilized thereon a captureantibody comprising SMA 4D8. Another embodiment comprises a releasemedium having releasably attached thereto a labeled antibody comprisingSMA 4D8 and a capture medium having immobilized thereon a captureantibody comprising SMA 3E8. Of course, further antibodies that show amoderate to high affinity for hCG-H and a reduced or lower affinity forother antigens also could be used. In various embodiments, the releasemedium and the capture medium can be in either non-overlapping fluidcommunication or overlapping communication.

The release medium, according to one embodiment of the presentinvention, includes a labeled conjugate comprising the detectable labeland a first binding member reactive with a first epitope of hCG-H and abiotinylated capturable component including a second binding memberreactive with a second epitope of hCG-H. Preferably, at least one of thefirst binding member and the second binding member exhibits a moderateto high affinity for hCG-H and is selectively or preferentially reactivewith hCG-H. As such, when a sample includes hCG-H, a sandwich complex isformed comprising the labeled conjugate, hCG-H, and the biotinylatedcapturable component. In such embodiments, the capture medium comprisesa capture site having immobilized thereon a capture component comprisingstreptavidin. In one such embodiment, only the first binding memberexhibits a moderate to high affinity for hCG-H and is selectively orpreferentially reactive with hCG-H. In yet another such embodiment, onlythe second binding member exhibits a moderate to high affinity for hCG-Hand is selectively ore preferentially reactive with hCG-H. In onealternative embodiment, both the first binding member and the secondbinding member exhibit a moderate to high affinity for hCG-H and areselectively or preferentially reactive with hCG-H.

As illustrated in FIG. 10, the device for selectively detecting hCG-H ina liquid sample, according to one embodiment, comprises a biphasicsubstrate 18 including a release medium 30 formed of a first materialand a capture medium 32 in fluid communication with the release mediumand formed of a second, different material. The release medium 30includes a labeled binding conjugate 26 comprising a binding memberreactive with a first epitope of hCG-H or hCG and a biotinylatedcapturable component 28 having a site reactive with a second epitope ofhCG-H or hCG, such that if hCG-H is present in the sample, a sandwichcomplex is formed comprising the labeled binding conjugate 26, hCG-H,and the biotinylated capturable component 28. Preferably, at least oneof the labeled binding conjugate 26 and the biotinylated capturablecomponent 28 includes a binding member that exhibits a moderate to highaffinity for hCG-H and is selectively or preferentially reactive withhCG-H. The capture medium 32 includes a capture site 34 for capturingthe complex, wherein the capture site includes a capture componentimmobilized thereon. The capture component can comprise streptavidin.Further, such embodiments can optionally include a control site 36. Incertain embodiments, the control site 36 can comprise either a negativeor positive control as discussed earlier.

According to embodiments including streptavidin as a capture component,the streptavidin used in the preparation of test devices according tothe invention preferably comprise a streptavidin solution that can beapplied to the test device, thereby immobilizing streptavidin on thesubstrate. The streptavidin in the solution can comprise a number ofpolymerized forms, such as dimeric, trimeric, tetrameric, or the like.While monomeric streptavidin can be present in the solution, thesolution preferably comprises a majority of polymerized streptavidin,the total content of any monomeric streptavidin in the solutioncomprising only a minority of the total content of the solution. Inspecific embodiments, the streptavidin solution comprises polymerizedstreptavidin in an amount such that the polymerized streptavidincomprises at least 50% by weight of the streptavidin solution.Preferably, the solution comprises at least about 55% by weight, atleast about 60% by weight, at least about 75% by weight, or at leastabout 90% by weight of polymerized streptavidin.

In one embodiment of the present invention, only the biotinylatedcapturable component comprises a binding member that exhibits a moderateto high affinity for hCG-H and is selectively or preferentially reactivewith hCG-H. In other embodiments, only the labeled conjugate comprises abinding member exhibiting a moderate to high affinity for hCG-H and isselectively or preferentially reactive with hCG-H. In one alternativeembodiment, both the biotinylated capturable component and the labeledconjugate comprise a binding member exhibiting a moderate to highaffinity for hCG-H and are selectively or preferentially reactive withhCG-H. For instance, one embodiment can comprise a labeled conjugateincluding a binding member exhibiting a moderate to high affinity ofhCG-H and being selectively or preferentially reactive with a firstepitope of hCG-H and the biotinylated capturable component can comprisesa different binding member that exhibits a moderate to high affinity forhCG-H and is selectively or preferentially reactive with a secondepitope of hCG-H.

According to certain embodiments, at least one of the biotinylatedcapturable component or the labeled antibody comprises SMA 3E8. In otherembodiment, at least one of the biotinylated capturable component or thelabeled antibody comprises SMA 4D8. Alternatively, one embodiment of theinvention comprises a release medium having releasably attached theretoa labeled antibody comprising SMA 3E8 and a biotinylated capturablecomponent comprising SMA 4D8, or vice versa. Again, further antibodiesthat show a moderate to high affinity for hCG-H and a reduced or loweraffinity for other antigens also could be used. The capture medium caninclude a capture component immobilized thereon. Preferably, the capturecomponent comprises streptavidin; more preferably polymerizedstreptavidin being at least about 100 kDa in size.

Indirect Detection

In another aspect, the present invention provides devices wherein thedetection is facilitated through selective removal of the hCG isoform.Such embodiments can particularly include a scavenger component (e.g.,antibody) that is selectively or preferentially reactive with regularhCG. Accordingly, the scavenger component can exhibit minimal, if any,reactivity with hCG-H. In various embodiments, the scavenger componentcan be located at any position between the location of sample depositand the capture site. Accordingly, the scavenger component effectively“subtracts out” the regular hCG from the sample, leaving primarily oronly hCG-H available for sandwich formation and ultimately colordevelopment at the capture site. As such, these embodiments allow forthe effective removal (e.g., by binding the regular hCG) of most (orall) regular hCG from the sample prior to contact with the capture site.With most or all of the regular hCG being effectively “subtracted out”of the sample, hCG-H will primarily be detected at the capture site.

In certain embodiments, the device includes a release medium comprisinga labeled conjugate including a detectable label and a first bindingmember that is reactive with a first epitope common to regular hCG andhCG-H while the capture medium comprises a capture site including asecond binding member that is reactive with a second epitope common toregular hCG and hCG-H. In one embodiment, the scavenger component can belocated at any position downstream from the sample deposit and up to andincluding a region including the labeled conjugate. In anotherembodiment, the scavenger component can be located between a regionincluding the labeled conjugate and a region including the capture site.In yet another embodiment, the scavenger component can be located at anyregion on the capture medium upstream from the capture site. Preferably,the scavenger component (e.g., antibody) exhibits a moderate to highaffinity for regular hCG and is selectively or preferentially reactivewith hCG.

As illustrated in FIG. 11, the device for detecting the presence orabsence of hCG-H in a liquid sample, according to one embodiment,comprises a biphasic substrate 18 wherein the biphasic substrate 18includes a release medium 30 formed of a first material and comprising alabeled conjugate 26 having a binding member reactive with a firstepitope common to hCG-H and regular hCG and a capture medium 32 in fluidcommunication with the release medium 30. The capture medium 32 can beformed of a second, different material, and include a capture site 34having immobilized thereon a member reactive with a second epitopecommon to hCG-H and regular hCG. Additionally, a detection deviceaccording such an embodiment includes a scavenger antibody 20 exhibitingsubstantially no reactivity with hCG-H. The scavenger antibody 20 cancomprise a binding member selectively or preferentially reactive withhCG, wherein the scavenger antibody 20 can be located between a depositlocation of the liquid sample and the capture site 34. The scavengerantibody can be releasably attached to the biphasic substrate 18 fromany location between a sample deposit location and the capture site 34.As such, if hCG-H is present in the sample, a complex is formedcomprising the labeled conjugate 26, hCG-H, and the immobilized reactivemember 34.

In certain alternative embodiments, one or both of the labeled conjugate26 and the immobilized binding member 34 can comprise a binding memberthat exhibits a moderate to high affinity for hCG-H and is selectivelyor preferentially reactive with hCG-H. In other embodiments, however,neither the labeled conjugate 26 nor the immobilized binding member 34comprise a binding member that exhibits a moderate to high affinity forhCG-H while also being selectively or preferentially reactive withhCG-H.

According to various embodiments, the scavenger component (e.g.,antibody) can be located downstream from the deposit location of theliquid sample and up to and including a region including the labeledconjugate. In other embodiments, the scavenger antibody can be locatedbetween a region including the labeled conjugate and a region includingthe capture site. Alternatively, the scavenger antibody can be locatedat any region on the capture medium upstream from the capture site.

In certain embodiments, the pregnancy device includes a release mediumcomprising a detectable label and a first binding member reactive with afirst epitope common to hCG and hCG-H and a biotinylated capturablecomponent comprising a second binding member reactive with a secondepitope common to hCG and hCG-H. Such devices also include a capturemedium including a capture site having immobilized thereon a capturecomponent comprising streptavidin. According to such embodiments, ascavenger component can be located downstream from the sample depositand up to and including a region including the labeled conjugate. Inanother embodiment, such devices can include a scavenger componentlocated downstream from a region including the labeled conjugate and upto and including a region including the biotinylated capturablecomponent. In yet another embodiment, the scavenger component can belocated at any region on the capture medium upstream from the capturesite. In such embodiments, the scavenger component effectively“subtracts out” the regular hCG from the sample, leaving primarily oronly hCG-H available for sandwich formation and ultimately colordevelopment at the capture site. With the effective removal of regularhCG from the sample (e.g., by binding with the scavenger component), anyhCG-H present in the sample forms a complex comprising the labeledconjugate, hCG-H, and the biotinylated capturable component.

As illustrated by FIG. 12, one embodiment of the invention comprises adevice which includes a biphasic substrate 18. The biphasic substrate 18can comprise a release medium 30 formed of a first material and acapture medium 32 in fluid communication with the release medium 30 andformed of a second, different material. The release medium 30 caninclude a labeled conjugate 26 comprising a binding member which isreactive with a first epitope common to hCG-H and hCG and a biotinylatedcapturable component 28 having a site reactive with a second epitopecommon to hCG-H or hCG. The embodiment illustrated by FIG. 12 includesthe scavenger component 20 located upstream of the labeled conjugate 26.The scavenger component (e.g., antibody) 20 is preferably selectively orpreferentially reactive with hCG. That is, the scavenger component 20preferably exhibits substantially no reactivity with hCG-H. Thescavenger component 20 can react with regular hCG present in the liquidsample to effectively “subtract out” regular hCG from the sample priorto the sample contacting the labeled conjugate 26. As such, the labeledconjugate will primarily bind to hCG-H and ultimately a complex isformed comprising the labeled conjugate 26, hCG-H, and the biotinylatedcapturable component 28. In these embodiments, the capture medium 32 cancomprise a capture site 34 for capturing the complex, wherein thecapture site 34 has immobilized thereon a capture component comprisingstreptavidin. In one preferred embodiment, the capture componentcomprises polymerized streptavidin, more preferably greater than 50% byweight of the polymerized streptavidin is at least about 100 kDa insize. Accordingly, the color development at the capture site 34 Variousembodiments can also include a control site 36 located on the capturemedium 32.

In certain alternative embodiments, one or both of the labeled conjugate26 and the biotinylated capturable component 28 can comprise a bindingmember that exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with hCG-H. In other embodiments,however, neither the labeled conjugate 26 nor the biotinylatedcapturable component 28 comprise a binding member that exhibits amoderate to high affinity for hCG-H while also being selectively orpreferentially reactive with hCG-H.

Ratiometric Detection

In another aspect, the present invention provides devices whereindetection is based on a ratiometric analysis. Such embodiments caninclude a mixture of binding members comprising a first group of bindingmembers that are preferably (but not necessarily) selectively orpreferentially reactive with an epitope of regular hCG and a secondgroup of binding members that exhibit a moderate to high affinity forhCG-H and are selectively or preferentially reactive with an epitope ofhCG-H. In certain embodiments, the first group of binding members thatare selectively or preferentially reactive with an epitope of regularhCG are located at a different location than the second group of bindingmembers that exhibit a moderate to high affinity for hCG-H and areselectively or preferentially reactive with an epitope of hCG-H. Thatis, each group of binding members can be striped independently of eachother if desired. In one preferred embodiment, the binding members thatare selectively or preferentially reactive with hCG-H comprise greaterthan 50% of the total number of binding members present in the mixture.

In various embodiments, the release medium includes different groups ofbinding antibodies (e.g., pooled together in a single stripe, stripedindependently of each other, or striped on top or each other) includinga first group preferably (but not necessarily) being selectively orpreferentially reactive with an epitope of regular hCG and a secondgroup that exhibits a moderate to high affinity for hCG-H and areselectively or preferentially reactive with an epitope of hCG-H.Alternatively, the capture site can include different groups of captureantibodies including a first group preferably (but not necessarily)selectively or preferentially reactive with an epitope of regular hCGand a second group that exhibits a moderate to high affinity for hCG-Hand are selectively or preferably reactive with an epitope of hCG-H. Ifdesired, both the release medium and the capture medium can include thedifferent groups of such antibodies (e.g., pooled together in a singlestripe, striped independently of each other, or striped on top or eachother).

In one preferred embodiment, the capture site comprises a mixture ofregular hCG and hCG-H specific antibodies, wherein about 50% to about98% of the antibodies are selectively or preferentially reactive withhCG-H and the remainder are selectively or preferentially reactive withregular hCG. Alternatively, the capture site can comprise about 70% toabout 95% of capture antibodies being selectively or preferentiallyreactive with hCG-H, or from about 85% to about 95%, or from about 90%to about 95%. In one alternative embodiment, the capture site comprisesabout 90% to about 99% of capture antibodies selectively orpreferentially reactive with hCG-H. In preferred embodiments, the hCG-Hantibody or antibodies also exhibit a moderate to high affinity forhCG-H.

Such embodiments can allow for appropriate visual signal strength to bepresent at the test line regardless of when the device is employedduring pregnancy. For instance, a user may choose to employ a pregnancytest device of the present invention later in pregnancy such as severalweeks beyond the day of the missed period when the levels of hCG-H arereduced and the level of regular hCG present is proportionally higher.The discrimination by the hCG-H selective or preferential antibody orantibodies can allow specific detection of low levels of hCG-H in thesample during the early days of pregnancy, while the presence of theregular hCG antibodies will ensure a pregnant result throughoutpregnancy. Thus, devices according to these embodiments can detect hCG-Hin liquid samples having either low levels or high levels of hCG-H.

Multiple Line Assay

In yet another aspect of the present invention, the device can comprisea multiple line assay device for selectively detecting hCG-H in a liquidsample is provided. The device includes a release medium formed of afirst material and comprising a detectable label and at least onebinding member that exhibits a moderate to high affinity for hCG-H andis selectively or preferentially reactive with an epitope of hCG-H.Devices according to such embodiments also include at least one bindingmember that is reactive with an epitope of regular hCG and a capturemedium in fluid communication with the release medium, wherein therelease medium is preferably formed of a second, different material. Thecapture medium can include a first capture site that binds hCG-H and asecond capture site that binds regular hCG. In addition to the twocapture sites, the capture medium can also include a control site toindicate that the device is working properly. Accordingly, oneembodiment of the present invention can have a capture medium with threelines or sites for possible color development. For instance, the secondcapture site that binds regular hCG can show the presence of total hCGindicating pregnancy. The first capture site can show the presence ofhCG-H indicating viability of the pregnancy and the control site willshow that the test is functional. As such, a viable pregnancy can bedetected as well as confirmation that the device is functional by visualcolor development of all three sites. That is, a user will see threecolored lines in the test window of the device.

As illustrated in FIG. 13 A-C, such embodiments can beneficially includea capture medium having separate capture sites within the window of thedevice, wherein one capture site is specific for regular hCG 100 and asecond capture site is specific for hCG-H 200. Optionally, a third linecomprising a control line 300 can be included so that a user can obtainthree independent results. Specifically, devices according to theseparticular embodiments of the invention provide a user indication oftest functionality (control line 300), pregnancy (detection of regularhCG 100), and the viability of the pregnancy (detection of hCG-H 200) inone test strip. For instance, FIG. 13A depicts test results conveyingthat the user is not pregnant and that the test device is workingproperly. FIG. 13B depicts test results conveying (1) that the user ispregnant; (2) the pregnancy is viable; and (3) that the device isfunctioning properly. FIG. 13C depicts test results for a user in whichconception has occurred as evidenced by the color development at thetest line 100, but that the pregnancy is likely not viable as evident bythe lack of color development at the hCG-H line 200. Further, the colordevelopment at the control line 300 indicates that the device is workingproperly. As such, the test results indicate the occurrence of an earlypregnancy loss, wherein the zygote will likely not successfully implantand proceed as a viable pregnancy.

In one embodiment, a multiple line assay device includes a releasemedium comprising a labeled conjugate including a detectable label andat least one binding member that is reactive with a first epitope ofhCG-H and a labeled conjugate comprising a detectable label and at leastone binding member that is reactive with a first epitope of regular hCG.Such devices also include a capture medium comprising a capture siteincluding a binding member that is reactive with a second epitope ofhCG-H and a separate capture site comprising a binding member that isreactive with a second epitope of regular hCG. In accordance with suchembodiments, at least one of the binding members preferably exhibits amoderate to high affinity for hCG-H and is selectively or preferentiallyreactive with an epitope of hCG-H. In one embodiment, at least one ofthe binding members reactive with an epitope of regular hCG ispreferably selectively or preferentially reactive with an epitope ofregular hCG.

In yet another embodiment, a multiple line assay device can include arelease medium comprising a labeled conjugate including a detectablelabel and at least one binding member that is reactive with an epitopecommon to both hCG-H and regular hCG. Further, the device can include acapture medium including a capture site comprising a binding member thatexhibits a moderate to high affinity for hCG-H and is selectively orpreferentially reactive with an epitope of hCG-H. The capture site cancomprise a binding member that is reactive with an epitope of regularhCG.

According to various embodiments of the present invention, the devicecomprises a biphasic substrate including a release medium formed of afirst material and a capture medium in fluid communication with therelease medium and formed of a second, different material. The releasemedium includes a first labeled conjugate comprising a binding memberthat exhibits a moderate to high affinity for hCG-H and is selectivelyor preferentially reactive with an epitope of hCG-H and a second labeledconjugate comprising a binding member selectively or preferentiallyreactive with a first epitope of regular hCG. The capture medium caninclude a first capture site comprising an immobilized member thatexhibits a moderate to high affinity for hCG-H and is selectively orpreferentially reactive with a second epitope of hCG-H, such that ifhCG-H is present in the sample, a sandwich complex is formed comprisingthe first labeled conjugate, hCG-H, and the first immobilized bindingmember. The capture medium preferably also includes a second capturesite comprising a second immobilized binding member reactive with asecond epitope of regular hCG, such that if hCG is present in thesample, a sandwich complex is formed comprising the second labeledconjugate, hCG, and the second immobilized binding member.

In yet another embodiment, the device can include a release mediumcomprising a labeled conjugate including a detectable label, at leastone binding member reactive with a first epitope of hCG-H, and at leastone binding member reactive with a first epitope of hCG. The releasemedium can also include a biotinylated capturable component comprising abinding member that exhibits a moderate to high affinity for hCG-H andis selectively or preferentially reactive with a second epitope ofhCG-H. Such embodiments can include a capture medium including a capturesite having immobilized thereon a capture component comprisingstreptavidin and a capture site comprising a binding member that isreactive with a second epitope of regular hCG.

In certain embodiments of the present invention, the device can includea release medium comprising a labeled conjugate including a detectablelabel, at least one binding member reactive with a first epitope ofhCG-H, and at least one binding member reactive with a first epitope ofregular hCG. The release medium can also include a biotinylatedcapturable component comprising a binding member that is selectively orpreferentially reactive with a second epitope of regular hCG. In suchembodiments, the capture medium comprises a capture site havingimmobilized thereon a capture component comprising streptavidin and acapture site comprising a binding member that is reactive with a secondepitope of hCG-H.

In certain embodiments, the release medium formed of a first materialcan include a labeled conjugate comprising a binding member reactivewith a first epitope of hCG-H and hCG (e.g., a common epitope of hCG-Hand hCG), and a capture medium in fluid communication with the releasemedium and formed of a second, different material. The capture mediumcan include a first capture site comprising an immobilized bindingmember that exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with a second epitope of hCG-H,such that if hCG-H is present in the sample, a sandwich complex isformed comprising the labeled conjugate, hCG-H, and the firstimmobilized binding member. The capture medium can also include a secondcapture site comprising an immobilized member selectively orpreferentially reactive with a second epitope of regular hCG, such thatif regular hCG is present in the sample, a sandwich complex is formedcomprising the second labeled conjugate, regular hCG, and the secondimmobilized binding member.

Multi-Strip Assay

In another aspect, the present invention provides a test device forselectively detecting hCG-H in a liquid sample which would allowmultiple results to be conveyed to the consumer in one test kit by wayof a multi-strip assay format. In such embodiments, two or moreindependent test strips share a common fluid path to convey independentresults. FIG. 14 illustrates one such embodiment, wherein the deviceincludes two strips, namely strip A 410 and strip B 440. The housing(not shown) of the pregnancy test device can be designed to accommodatetwo or more test strips. In a preferred embodiment, one of the strips isspecific for regular hCG (e.g., Strip A 410 in FIG. 14) and the otherstrip is specific for hCG-H (e.g., Strip B 440 in FIG. 14). The stripscan share a common sampling component 400, such as a urine wick, whichwould allow sample to be transferred to both strips simultaneously. Uponusing the device, the consumer can preferably view two independentresults windows, depicted as dashed lines on FIG. 14, to obtain theresults. For instance, the appearance of a test line 100 in one windowfor strip A 410 on FIG. 14 can convey that the user is pregnant whilethe appearance of an hCG-H line 200 in an independent window for strip B440 can convey that the pregnancy is viable, and likely at low risk forearly pregnancy loss.

As depicted in FIG. 15, in another variation the capture mediums (e.g.,nitrocellulose regions) of the two or more strips can be joined to acommon release medium 480 and share a common fluid path 400 to conveytwo independent results. Again, the housing of the pregnancy test devicecan easily be designed to accommodate the larger unified test strip. Inaccordance with such embodiments, one portion of the device can beconstructed to specifically detect regular hCG (Side A 410 in FIG. 15),and another portion can be constructed to specifically detect hCG-H(Side B 440 in FIG. 15). A common fluid path 400 (e.g., urine wick) cantransfer sample to the release medium, whereby a labeling antibody cantravel up both portions of the strip (e.g., side A 410 and side B 440 inFIG. 15). In certain embodiments, the individual portions (e.g., sides Aand B) of the test strip can contain independent biotinylatedantibodies; wherein one of the biotinylated antibodies specificallybinds with an epitope of regular hCG 125 and the other biotinylatedantibody specifically binds an epitope of hCG-H 225. Upon running thedevice a user can view two independent results windows, depicted asdashed lines on FIG. 15, to obtain the results. The appearance of colordevelopment at a test line in one window (e.g., side A of FIG. 15) canconvey that the user is pregnant (i.e., color development at 150). Theappearance of color development at an hCG-H line in an independentwindow (e.g., Side B of FIG. 15) can convey that the pregnancy is viable(i.e., color development at 250), and likely at low risk for earlypregnancy loss.

Devices according to certain embodiments of the present invention canhave a common fluid path for receiving and distributing the liquidsample and at least one release medium in fluid communication with thecommon fluid path. The at least one release medium can be formed of afirst material and include a detectable label. Moreover, suchembodiments can include a first capture medium in fluid communicationwith the at least one release medium and formed of a second material,wherein the first capture medium comprises a capture site that directlyor indirectly selectively binds hCG-H. Devices according to theseparticular embodiments can include a second capture medium in fluidcommunication with the at least one release medium and formed of a thirdmaterial. The second capture medium can comprise a capture site thatdirectly or indirectly binds regular hCG. In one embodiment, the atleast one release medium comprises a first release medium including alabeled conjugate comprising a detectable label and a binding memberthat is reactive with a first epitope of regular hCG-H and a secondrelease medium comprising a labeled conjugate including a detectablelabel and a binding member that is reactive with a first epitope ofregular hCG. Devices according to such embodiments include a firstcapture medium being in fluid communication with the first releasemedium; wherein the first capture medium can comprise a capture siteincluding a binding member that exhibits a moderate to high affinity forhCG-H and is selectively or preferentially reactive with a secondepitope of hCG-H. Such embodiments can include a second capture mediumin fluid communication with the second release medium, wherein thesecond capture medium can comprise a capture site including a bindingmember that is reactive with a second epitope of regular hCG.

In certain embodiments, the device comprises a first biphasic substrateincluding (i) a first release medium formed of a first material andcomprising a first labeled conjugate comprising a binding member thatexhibits a moderate to high affinity for hCG-H and is selectively orpreferentially reactive with a first epitope of hCG-H and (ii) a firstcapture medium in fluid communication with the first release medium andformed of a second, different material. The first capture mediumcomprises a first capture site comprising a first immobilized memberpredominantly reactive with a second epitope of hCG-H, such that ifhCG-H is present in the sample, a sandwich complex is formed comprisingthe first labeled conjugate, hCG-H, and the first immobilized bindingmember. The device can also include a second biphasic substratecomprising (i) a second release medium formed of a first material andcomprising a second labeled conjugate comprising a binding memberselectively or preferentially reactive with a first epitope of regularhCG and (ii) a second capture medium in fluid communication with thesecond release medium and formed of a second, different material. Thesecond capture medium can include a second capture site comprising asecond immobilized binding member reactive with a second epitope ofregular hCG, such that if regular hCG is present in the sample, asandwich complex is formed comprising the second labeled conjugate,regular hCG, and the second immobilized binding member. Devicesaccording to such embodiments can include a liquid sample deposit sitebeing in fluid communication with both the first biphasic substrate andthe second biphasic substrate. In certain embodiments of the presentinvention, the device can comprise a multi-strip assay device includinga common release medium.

In yet another embodiment, a multi-strip device can include one or morerelease mediums. For instance, such a device can included a firstrelease medium comprising a labeled conjugate including a detectablelabel and a binding member that is reactive with a first epitope ofhCG-H and a biotinylated capturable component comprising a bindingmember that exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with a second epitope of hCG-H.Further, a second release medium can be provided, wherein the secondrelease medium can comprise a labeled conjugate including a detectablelabel and a binding member that is reactive with a first epitope ofregular hCG. These particular devices can include multiple capturemediums; wherein each capture medium can be in fluid communication witha respective release medium. For instance, such embodiments can includea first capture medium being in fluid communication with the firstrelease medium. The first capture medium can comprise a capture sitehaving immobilized thereon a capture component comprising monomeric orpolymerized streptavidin. A second capture medium can also be providedas to be in fluid communication with the second release medium. Thesecond capture medium can comprise a capture site including a bindingmember that is reactive with a second epitope of regular hCG. In oneparticular embodiment, the device can comprise a single release mediumin fluid communication with both a first capture medium and a secondcapture medium. The release medium can comprise a labeled conjugateincluding a detectable label and a binding member that is reactive witha first epitope of hCG-H and a first epitope of regular hCG.

In yet another embodiment, the first capture medium can comprise acapture site including a binding member that exhibits a moderate to highaffinity for hCG-H and is selectively or preferentially reactive with asecond epitope of hCG-H. Further, the second capture medium can comprisea capture site including a binding member that is reactive with a secondepitope of regular hCG. In another embodiment, the device can include arelease medium comprising a biotinylated capturable component. Thebiotinylated capturable component preferably comprises a binding memberthat is selectively or preferentially reactive with a second epitope ofhCG-H. As such, the first capture medium can comprise a capture sitehaving immobilized thereon a capture component comprising monomeric orpolymerized streptavidin.

In various embodiments, the device for detecting the presence of bothregular hCG and hCG-H in a liquid sample can comprise a first biphasicsubstrate including a first release medium formed of a first material.The first release material can include a first labeled conjugatecomprising a binding member that exhibits a moderate to high affinityfor hCG-H and is selectively ore preferentially reactive with a firstepitope of hCG-H and a first biotinylated capturable component having asite that exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with a second epitope of hCG-H,such that if hCG-H is present in the sample, a first sandwich complex isformed comprising the first labeled conjugate, hCG-H, and the firstbiotinylated capturable component. A first capture medium in fluidcommunication with the first release medium and formed of a second,different material, can be included. The first capture medium comprisesa first capture site, for capturing the first complex, including animmobilized capture component comprising monomeric or polymerizedstreptavidin. In such embodiments, one or more additional biphasicsubstrates can be included for the detection of various hCG isoforms. Inone preferred embodiment, the device includes a second biphasicsubstrate comprising a second release medium formed of a first materialand comprising a second labeled conjugate comprising a binding memberreactive with a first epitope of regular hCG and a second biotinylatedcapturable component having a site reactive with a second epitope ofregular hCG, such that if regular hCG is present in the sample, a secondcomplex is formed comprising the second labeled conjugate, regular hCG,and the second biotinylated capturable component. In a more preferredembodiment, at least one of the second labeled conjugate and the secondbiotinylated capturable component is selectively or preferentiallyreactive with regular hCG. Further, a device according to suchembodiments includes a second capture medium in fluid communication withthe second release medium and formed of a second, different material.The second capture medium comprises a second capture site for capturingthe second complex including an immobilized capture component comprisingmonomeric or polymerized streptavidin. According to embodiments of thepresent invention, a deposit site for a liquid sample should preferablybe in fluid communication with both the first biphasic substrate and thesecond biphasic substrate. In various embodiments, the device comprisesa common release medium.

Viability of a Pregnancy

In another aspect, the present invention provides a method forevaluating the viability of a pregnancy. Such methods, according tovarious embodiments, allow a user to evaluate the viability of apregnancy, wherein some methods comprise providing a test deviceaccording to any of the embodiments described herein and applying aliquid sample to the device. Preferably, the liquid sample potentiallyincludes one or both regular hCG and hCG-H. Further, such methodsinclude detecting the presence or lack thereof of hCG-H in the liquidsample. The detected presence of hCG-H indicates a high probability of asuccessful (or viable) pregnancy. In certain embodiments according tothe present invention, the detection of hCG-H with test devicesaccording to certain embodiments of the present invention indicates aprobability of successful implantation and therefore a viable pregnancyof at least 50% (e.g., 50%-100%, 60%-90%, 70%-90%, etc.), at least 60%(60%-100%, 70%-90%, etc.), at least 70% (e.g., 70%-100%, 80%-100%,80%-90%, etc.), at least 80% (80%-100%, 90%-95%, etc.), at least 90%(e.g., 90%-100%, 95%-99%), or at least 95% (e.g., 95%-100%).

In a study conducted using urine samples, term pregnancies had a meanhCG-H concentration of 6 mIU/mL on the day of implantation (compared to2 mIU/mL hCG-H in Early Pregnancy Loss). See Sasaki et al. 2008:“Hyperglycosylated hCG and the source of Pregnancy Failures”; Fertilityand Sterility, Volume 89, Issue 6, Pages 1781-1786. In this same study,in early pregnancy a greater than 50% proportion of hCG-H (out of totalhCG) was required in order to proceed to term.

Example

A series of experiments were conducted to assess the relative affinitiesof the SMA4D8 and SMA3E8 antibodies. Both antibodies were analyzed byBiacore (GE Healthcare) and Enzyme-Linked ImmunoSorbent Assay (“ELISA”).

Biacore is an instrument that helps analyze antibody—antigeninteractions in terms of specificity, kinetics and affinity. Biacoreexploits the phenomena of Surface Plasmon Resonance (“SPR”). Biacoreallows the label free study of biomolecular interactions. Morespecifically, an antigen is coated onto a Biacore chip, and the relativeaffinities of different antibodies are probed in equilibrium. Forinstance, an antigen can be attached to the surface of a chip and asample containing an antibody can be passed over the surface of the chipto evaluate the interaction between the antigen and antibody.

The ELISA is a multiwell plate assay that utilizes antibodies to detectthe presence of an antigen in a sample. The ELISA assay allows ‘antigenfingerprinting’ to be performed. Antigen fingerprinting gives a snapshotof the differential recognition of antigens by a particular antibody.This method also allows one to determine the differential recognition ofan antibody to various isoforms/subunits/fragments of a large molecule.This biochemical technique is used mainly in immunology to detect thepresence of an antibody or an antigen in a sample. In simple terms, inELISA an antigen is affixed to a surface, and then a specific antibodyis washed over the surface so that it can bind to the antigen. Thisantibody is linked to an enzyme, and in the final step a substrate isadded that the enzyme can convert to some detectable signal. Thus in thecase of fluorescence ELISA, when light is shone upon the sample, anyantigen/antibody complexes will fluoresce so that the amount of antigenin the sample can be measured.

Performing an ELISA involves at least one antibody and an antigen. Theantigen is immobilized on a solid support (usually a polystyrenemicrotiter plate) either non-specifically (via adsorption to thesurface) or specifically (via capture by another antibody specific tothe same antigen, in a “sandwich” ELISA). After the antigen isimmobilized the detection antibody is added, forming a complex with theantigen. The detection antibody can be covalently linked to an enzyme,or can itself be detected by a secondary antibody which is linked to anenzyme through bioconjugation. Between each step the plate is typicallywashed with a mild detergent solution to remove any proteins orantibodies that are not specifically bound. After the final wash stepthe plate is developed by adding an enzymatic substrate to produce avisible signal, which indicates the presence or quantity of antigen inthe sample.

SMA 4D8 Biacore Affinity Analysis

The Biacore instrument was utilized to study the biomolecularinteractions between SMA 4D8 and particular antigens of interest. Assuch, the following antigens were coated onto a Biacore chip and SMA 4D8was separately passed over the surface of the chip: (1) normallyglycosylated hCG (4^(th) IS, NIBSC); (2) free β hCG (NIBSC); (3)recombinant hCG (Sigma); (4) JEG-3 derived purified hCG-H; and (5)Luteinizing Hormone (LH) (NIBSC). After each injection of antibody overthe chip, the Biacore chip was regenerated by passage of glycine at pH1.5. Thus, the antigen concentration is constant for each injection ofantibody. The concentrations used for the antibodies in each experimentvary (i.e., 1.0 to 12.5 μg/ml). The reason for this is that the antigenconcentration is constant but the response to an antigen for eachantibody differs and the aim was to provide a good range of bindingcurves from little binding to near saturation. The constants calculatedfor individual injections have been averaged. The calculated constantsare: (1) KA−affinity at equilibrium; (2) KD−dissociation equilibriumconstant; (3) ka−rate of complex formation; and (4) kd−rate ofdissociation. These values are provided in Table 1 below. The Biacoresensorgrams for normally glycosylated hCG, free β hCG, recombinant hCG,and JEG-3 hCG-H are provided in FIGS. 16, 17, 18, and 19, respectively.

TABLE 1 KA KD Ka Kd Affinity Stability hCG-H (JEG3) 1.65e¹⁰ 1.06e⁻¹⁰2.73e⁵ 2.98e⁻⁵ Good Stable Normally 4.57e⁹  2.45e⁻¹⁰ 2.79e⁵ 6.47e⁻⁵Moderate Some Glycosylated disso- hCG ciation Recombinant 1.21e⁸ 1.63e⁻⁹  1.09e⁵ 9.45e⁻⁴ Low Unstable hCG Free beta-hCG 3.45e⁸  3.26e⁻⁹ 1.31e⁵ 3.80e⁻⁴ Low Unstable LH — — — — — No Binding

As illustrated by the sensorgrams and the results summarized in Table 1,4D8 displayed highest affinity for hCG-H and moderate affinity fornormally glycosylated hCG with no LH cross-reactivity. As such, theepitope recognized by 4D8 may be influenced by glycosylation as it bindsto recombinant hCG with a significantly reduced affinity compared tonormally glycosylated hCG and hCG-H. Overall, the 4D8 Biacore resultsindicate a high affinity for hCG-H, a moderate affinity for normallyglycosylated hCG, little affinity for recombinant hCG and no binding toLH.

SMA 4D8 ELISA Analysis

A sandwich ELISA assay was utilized to help determine the differentialrecognition of various antigens by SMA 4D8 when presented by anotherantibody. Accordingly, an ‘antigen fingerprinting’ was performed toprovide a snapshot of the differential recognition of antigens by SMA4D8 when presented by the mouse monoclonal antibody 11D6-2B10 coated onthe microtiter plate. Additionally, the relative specificities for thedifferent presented antigens were illustrated by comparisons made inparallel. The results of the ELISA analysis are summarized in FIG. 20.All results in FIG. 20 were normalized to 100% choriocarcinoma (hCG-H)binding in order to allow an accurate comparison of antigenspecificities. FIG. 20 shows that SMA 4D8 exhibited a high specificityfor choriocarcinoma derived hCG-H, a high specifity for Early PregnancyUrine (EPU) derived hCG-H, moderate specifity for normally glycosylated(intact) hCG, moderate specifity for recombinant hCG, virtually norecognition for LH and virtually no recognition for Late Pregnancy Urine(LPU) derived hCG.

SMA 3E8 Biacore Affinity Analysis

The Biacore instrument was also utilized to study the biomolecularinteractions between SMA 3E8 and particular antigens of interest. Assuch, the following antigens were coated onto a Biacore chip and SMA 3EAseparately passed over the surface of the chip: (1) normallyglycosylated hCG (4^(th) IS, NIBSC); (2) free β hCG (NIBSC); (3)recombinant hCG (Sigma); (4) JEG-3 derived purified hCG-H; and (5)Luteinizing Hormone (LH) (NIBSC). After each injection of antibody overthe chip, the Biacore chip was regenerated by passage of glycine at pH1.5. Thus, the antigen concentration is constant for each injection ofantibody. The concentrations used for the antibodies in each experimentvary (i.e., 1.0 to 12.5 μg/ml). The reason for this is that the antigenconcentration is constant but the response to an antigen for eachantibody differs and the aim was to provide a good range of bindingcurves from little binding to near saturation. The constants calculatedfor individual injections have been averaged. The calculated constantsare: (1) KA−affinity at equilibrium; (2) KD−dissociation equilibriumconstant; (3) ka−rate of complex formation; and (4) kd−rate ofdissociation. These values are provided in Table 2 below. Thesensorgrams for normally glycosylated hCG, free β hCG, recombinant hCG,and JEG-3 hCG-H are provided in FIGS. 21, 22, 23, and 24, respectively.

TABLE 2 KA KD Ka Kd Affinity Stability hCG-H (JEG3) 2.27e⁹ 6.52e⁻¹⁰2.52e⁵ 1.23e⁻⁴  Moderate Some disso- ciation Normally 6.13e¹⁰ 5.85e⁻¹⁰1.47e⁵ 4.92e⁻⁵  Moderate Reason- Glycosylated able hCG Recombinant8.33e⁸ 3.70e⁻⁹  1.50e⁵ 4.165e⁻⁴ Low Unstable hCG Free beta-hCG 4.35e⁹2.99e⁻¹⁰ 5.92e⁵ 1.44e⁻⁴  Moderate Some disso- ciation LH 1.89e⁷ 6.49e⁻⁸ 1.59e⁵ 9.12e⁻³  Low Rapid disso- ciation

Overall, as illustrated by the sensorgrams and the results summarized inTable 2, 3E8 displayed a moderate affinity for hCG-H. 3E8 also displayeda moderate affinity for normally glycosylated hCG and free beta-hCG, andlow affinity for recombinant hCG and LH.

SMA 3E8 ELISA Analysis

A sandwich ELISA assay was utilized to help determine the differentialrecognition of various antigens by SMA 3E8 when presented by anotherantibody. Accordingly, an ‘antigen fingerprinting’ was performed toprovide a snapshot of the differential recognition of antigens by SMA3E8 when presented by the mouse monoclonal antibody 11D6-2B10 coatedonto the microtiter plate. Additionally, the relative specificities forthe different presented antigens were illustrated by comparisons made inparallel. The results of the ELISA analysis are summarized in FIG. 25.All results in FIG. 25 were normalized to 100% choriocarcinoma (hCG-H)binding in order to allow an accurate comparison of antigenspecificities. FIG. 25 shows that SMA 3E8 displays a high specifity forchoriocarcinoma derived hCG-H, a high specifity for Early PregnancyUrine (EPU) derived hCG-H, a moderate specifity for normallyglycosylated hCG, a moderate specifity for recombinant hCG, a lowspecifity for LH, a high specifity for Late Pregnancy Urine (LPU)derived hCG, and a high specifity for beta core fragment.

B152 Biacore Affinity Analysis

To provide a comparison the embodiments of the present invention with anantibody known for exhibiting specific recognition of hCG-H, the Biacoreinstrument was also utilized to study the biomolecular interactionsbetween B152 and particular antigens of interest. As such, the followingantigens were coated onto a Biacore chip and B152 separately passed overthe surface of the chip: (1) normally glycosylated hCG (4^(th) IS,NIBSC); (2) free β hCG (NIBSC); (3) recombinant hCG (Sigma); (4) JEG-3derived purified hCG-H; and (5) Luteinizing Hormone (LH) (NIBSC). Aftereach injection of antibody over the chip, the Biacore chip wasregenerated by passage of glycine at pH 1.5. Thus, the antigenconcentration is constant for each injection of antibody. Theconcentrations used for the antibodies in each experiment vary. Thereason for this is that the antigen concentration is constant but theresponse to an antigen for each antibody differs and the aim was toprovide a good range of binding curves from little binding to nearsaturation. The constants calculated for individual injections have beenaveraged. The calculated constants are: (1) KA−affinity at equilibrium;(2) KD−dissociation equilibrium constant; (3) ka−rate of complexformation; and (4) kd−rate of dissociation. These values are provided inTable 3 below. The sensorgrams for normally glycosylated hCG, free 13hCG, recombinant hCG, and JEG-3 hCG-H are provided in FIGS. 26, 27, 28,and 29, respectively.

TABLE 3 KA KD Ka Kd Affinity Stability hCG-H (JEG3) 6.54e⁸ 8.32e⁻⁹1.39e⁴ 8.31e⁻⁵ Low Unstable Normally 2.14e⁶ 1.72e⁻⁷ 1.93e³ 1.04e⁻³Negligible Unstable Glycosylated hCG Recombinant 5.05e⁷ 2.90e⁻⁸ 2.20e⁴4.04e⁻⁴ Low Unstable hCG Free beta-hCG 1.04e⁸ 2.14e⁻⁸ 2.31e⁵ 1.43e⁻³ LowUnstable LH — — — — — No Binding

Overall, as illustrated by the sensorgrams and the results summarized inTable 3, B152 exhibited a low affinity for hCG-H. The 7 injectionsillustrated by the sensograms on FIG. 29 show that concentration rangesbetween 20-100 micrograms/ml gave a response of 60-223 RU. Furthermore,at equilibrium the KA value of 6.54e⁸ and KD value of 8.32e⁻⁹ show thatB152 has a low affinity for hCG-H. For comparison, the SMA 3E8 exhibiteda KA value of 2.27e⁹ and a KD value of 6.52e⁻¹⁰ while SMA 4D8 exhibiteda KA value of 1.65e¹⁰ and a KD value of 1.06e⁻¹⁰. Furthermore, the B152concentration necessary (i.e., 100 micrograms/ml) to generate a responseof 223 RU was about 50 to 100 times greater than that of SMA 3E8 and SMA4D8. That is, to achieve a similar response to SMA 3E8 and SMA 4D8 theB152 concentration must be present at a concentration roughly a 100times greater.

Comparison of Traditional Devices with Devices including an AntibodyExhibiting a Moderate to High Affinity for hCG-H

As previously mentioned, FIGS. 2A-2C show the relative bindingaffinities for three antibody pairs (i.e., 3 separate devices eachhaving a unique pair of antibodies) in which one of the binding membersexhibits a moderate to high affinity for hCG-H in comparison to twoproduction devices that are commercially available. FIGS. 2A-2C eachillustrate the improvement in differential discrimination ofhyperglycosylated hCG and recombinant hCG. In addition to comparing therelative affinities of these particular antibody pairs, their respectiveintensity of color development was also compared to the currentproduction devices. As shown in FIG. 30, the color intensity (i.e.,G//Dens—a measure of color intensity) of the three antibody pairs (i.e.,CCFO1/3E8, CCFO1/4D8, and 11D6-2B10/4D8) were comparable to more intensethan the FR1 devices (i.e., First Response Early Result pregnancy testdevices from Church & Dwight Co., Inc.) compared to FR2 and FR1 devices.The FR2 devices (utilizing polymerized streptavidin) exhibited the bestresults. In certain embodiments according to the present invention,however, polymerized streptavidin can be utilized as the capturecomponent.

Additionally, clinical urine sample testing was conducted for devicesincluding these three particular antibody pairs (i.e., CCFO1/3E8,CCFO1/4D8, and 11D6-2B10/4D8). Once again, the results of these deviceswere compared to those of the current production devices. First, testingof hCG free urine samples of differing non-surge LH values (i.e., 0-60mIU/ml) were conducted. As shown in FIG. 31, each of the devicescorrectly provided a “negative” result for all urine samples. Thus,devices including the CCFO1/3E8, CCFO1/4D8, and 11D6-2B10/4D8 antibodypairs desirably did not provide a single “false positive”. Additionaltesting was conducted on hCG free urine samples of differingconcentrations of LH surge samples (i.e., >130 mIU/ml). As shown in FIG.32, each of the devices correctly provided a “negative” result for allurine samples. Again, devices including the CCFO1/3E8, CCFO1/4D8, and11D6-2B10/4D8 antibody pairs desirably did not provide a single “falsepositive”.

A large panel (200) of peri- and post-menopausal urine samples withvarying levels of pituitary hCG were collected for another study. Toevaluate the hCG-H detection of the poorly glycosylated pituitaryderived hCG, 10 samples containing pituitary hCG (2.4 to 28.5 mIU) wereselected form the larger sample set. As shown in FIG. 33, the FR2devices incorrectly provided a positive result on seven occasions. Incontrast, all devices for selectively detecting hCG-H correctly provideda negative results except for a single 11D6-2B10/4D8 device (testedagainst 28 mIU/ml of pituitary hCG). Thus, the devices for selectivelydetecting hCG-H provide a significant reduction in the detection ofpituitary hCG when compared to production devices that assay total hCG.

Further testing of these particular devices for selectively detectinghCG-H was conducted on a two sets (Set A and Set B) of wellcharacterized clinical urine samples from early pregnancy. The resultsof Set A are shown in FIG. 34 and the results of Set B are shown in FIG.35. As shown in FIGS. 34 and 35, each of the devices for selectivelydetecting hCG-H showed similar results as the production devices interms of giving a positive result prior to the day of an expectedmenstrual period (i.e., EMP on the FIGS). Beneficially, the11D6-2B10/4D8 test devices provided positive results on at least dayEMP-5 (i.e., 5 days prior to an expected menstrual period). Such anearly detection of pregnancy is equal to or better than the resultsobtained by the FR1 devices.

Given the abundance of literature which contends that hCG-H is importantfor successful implantation to occur, a high proportion of earlypregnancy loss (EPL) samples should theoretically exhibit low levels ofhCG-H. To assess this, four cycles of women who suffered EPL werecharacterized and tested with both production and each of theseparticular devices for selectively detecting hCG-H (i.e., CCFO1/3E8,CCFO1/4D8, and 11D6-2B10/4D8). The results of this testing are providedin FIG. 36. As shown in FIG. 36, subjects THME and BRDA receivedpositive results with the FR2 devices (based on total hCG assay) andpossibly positive results with the FR1 devices (based on total hCGassay). The particular devices for selectively detecting hCG-H (i.e.,CCFO1/3E8, CCFO1/4D8, and 11D6-2B10/4D8), however, all correctlyprovided negative results due to the absence of hCG-H in the sampleswhen tested. Beneficially, the 11D6-2B10/4D8 devices consistently gavenegative results for every sample.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

1. A device for selectively detecting hyperglycosylated human chorionicgonadotropin (hCG-H) in a liquid sample deposited directly or indirectlyon a proximal portion of the device for transport to a distal portion ofthe device, wherein the device comprises: A) a release medium formed ofa first material and comprising a detectable label; and B) a capturemedium in fluid communication with the release medium and formed of asecond, different material, the capture medium comprising a capturesite; wherein at least one of the release medium and the capture mediumcomprises a binding member that exhibits a moderate to high affinity forhCG-H and is selectively or preferentially reactive with hCG-H.
 2. Thedevice of claim 1, wherein: A) the release medium comprises a labeledconjugate comprising the detectable label and a first binding memberthat is reactive with at least one of an epitope of hCG-H or an epitopeof human chorionic gonadotropin (hCG); and B) the capture mediumcomprises a capture site comprising a second binding member immobilizedon the capture medium, said second binding member is reactive with atleast one of an epitope of hCG-H or an epitope of hCG; wherein at leastone of the first binding member and the second binding member exhibits amoderate to high affinity for hCG-H and is selectively or preferentiallyreactive with hCG-H.
 3. The device of claim 2, wherein only the firstbinding member exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with hCG-H.
 4. The device ofclaim 2, wherein only the second binding member exhibits a moderate tohigh affinity for hCG-H and is selectively or preferentially reactivewith hCG-H.
 5. The device of claim 2, wherein both the first bindingmember and the second binding member exhibit a moderate to high affinityfor hCG-H and are selectively or preferentially reactive with hCG-H. 6.The device of claim 2, wherein: A) the release medium comprises: i) alabeled conjugate comprising the detectable label and a first bindingmember reactive with a first epitope of hCG-H; and ii) a capturablecomponent comprising a second binding member reactive with a secondepitope of hCG-H, at least one of the first binding member and thesecond binding member exhibits a moderate to high affinity for hCG-H andbeing selectively or preferentially reactive with hCG-H, such that inthe presence of hCG-H in the sample, a complex is formed comprising thelabeled conjugate, hCG-H, and the capturable component; and B) thecapture medium comprises a capture site having immobilized thereon acapture component having a binding affinity for the capturablecomponent.
 7. The device of claim 6, wherein the capturable componentcomprises a biotinylated capturable component and the capture componentcomprises streptavidin.
 8. The device of claim 6, wherein only the firstbinding member exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with hCG-H.
 9. The device ofclaim 6, wherein only the second binding member exhibits a moderate tohigh affinity for hCG-H and is selectively or preferentially reactivewith hCG-H.
 10. The device of claim 6, wherein both the first bindingmember and the second binding member exhibit a moderate to high affinityfor hCG-H and are selectively or preferentially reactive with hCG-H. 11.A device for selectively detecting hyperglycosylated human chorionicgonadotropin (hCG-H) in a liquid sample deposited directly or indirectlyon a proximal portion of the device for transport to a distal portion ofthe device, wherein the device comprises: A) a release medium formed ofa first material and comprising a detectable label; B) a capture mediumin fluid communication with the release medium and formed of a second,different material, the capture medium comprising a capture site; and C)a scavenger component that is selectively or preferentially reactivewith regular hCG, the scavenger component being located between alocation of sample deposit and the capture site; wherein at least one ofthe release medium and the capture medium comprises a binding memberthat is reactive with hCG-H.
 12. The device of claim 11, wherein: A) therelease medium comprises a labeled conjugate comprising the detectablelabel and a first binding member that is reactive with a first epitopeof hCG-H; and B) the capture medium comprises a capture site comprisinga second binding member that is reactive with a second epitope of hCG-H.12. The device of claim 12, wherein at least one of the first bindingmember and the second binding member exhibits a moderate to highaffinity for hCG-H and is selectively or preferentially reactive withhCG-H.
 13. The device of claim 12, wherein the scavenger component islocated downstream from the sample deposit and up to and including aregion including the labeled conjugate.
 14. The device of claim 12,wherein the scavenger component is located between a region includingthe labeled conjugate and a region including the capture site.
 15. Thedevice of claim 12, wherein the scavenger component is located at anyregion on the capture medium upstream from the capture site.
 16. Thedevice of claim 11, wherein: A) the release medium comprises: i) thedetectable label and a first binding member reactive with a firstepitope of hCG-H; and ii) a capturable component comprising a secondbinding member reactive with a second epitope of hCG-H, such that in thepresence of hCG-H in the sample, a complex is formed comprising thelabeled conjugate, hCG-H, and the capturable component; and B) thecapture medium comprises a capture site having immobilized thereon acapture component having a binding affinity for the capturablecomponent.
 17. The device of claim 16, wherein the capturable componentcomprises a biotinylated capturable component and the capture componentcomprises streptavidin.
 18. The device of claim 16, wherein at least oneof the first binding member and the second binding member exhibits amoderate to high affinity for hCG-H and is selectively or preferentiallyreactive with hCG-H.
 19. The device of claim 16, wherein the scavengercomponent is located downstream from the sample deposit and up to andincluding a region including the labeled conjugate.
 20. The device ofclaim 16, wherein the scavenger component is located between a regionincluding the labeled conjugate and a region including the capture site.21. The device of claim 16, wherein the scavenger component is locatedat any region on the capture medium upstream from the capture site. 22.A device for selectively detecting hyperglycosylated human chorionicgonadotropin (hCG-H) in a liquid sample deposited directly or indirectlyon a proximal portion of the device for transport to a distal portion ofthe device, wherein the device comprises: A) a release medium formed ofa first material and comprising, (i) a first group of labeled conjugatescomprising a detectable label and a binding member that is selectivelyor preferentially reactive with an epitope of regular hCG; and (ii) asecond group of labeled conjugates comprising a detectable label and abinding member is selectively or preferentially reactive with an epitopeof hCG-H and exhibits a moderate to high affinity for hCG-H; and B) acapture medium in fluid communication with the release medium and formedof a second, different material, the capture medium comprising a capturesite having immobilized thereon at least one group of binding members;wherein the binding members that are selectively or preferentiallyreactive with hCG-H comprise greater than 50% of the total number ofbinding members present on the release medium.
 23. The device of claim22, wherein the first and second groups of labeled conjugates areprovided as a mixture.
 24. The device of claim 22, wherein the firstgroup of labeled conjugates is located at a different location on therelease medium than the second group of labeled conjugates.
 25. A devicefor selectively detecting hyperglycosylated human chorionic gonadotropin(hCG-H) in a liquid sample deposited on a proximal portion of the devicefor transport to a distal portion of the device, wherein the devicecomprises: A) a release medium formed of a first material and comprisinga detectable label; B) at least one binding member that exhibits amoderate to high affinity for hCG-H and is selectively or preferentiallyreactive with an epitope of hCG-H; C) at least one binding member thatis reactive with an epitope of regular hCG; and D) a capture medium influid communication with the release medium and formed of a second,different material, the capture medium comprising: (i) a first capturesite that binds hCG-H; and (ii) a second capture site that binds regularhCG.
 26. The device of claim 25, wherein the at least one binding memberreactive with an epitope of regular hCG is selectively or preferentiallyreactive with an epitope of regular hCG.
 27. The device of claim 25,wherein: A) the release medium comprises: i) a labeled conjugatecomprising a detectable label and at least one binding member that isreactive with a epitope common to regular hCG and hCG-H; and B) thecapture medium comprises: i) a capture site comprising a binding memberthat is reactive with a second epitope of hCG-H; and ii) a capture sitecomprising a binding member that is reactive with a second epitope ofregular hCG; wherein at least one of the binding members reactive withan epitope of hCG-H exhibits a moderate to high affinity for hCG-H andis selectively or preferentially reactive with hCG-H.
 28. The device ofclaim 27, wherein at least one of the binding members reactive with anepitope of regular hCG is selectively or preferentially reactive with anepitope of regular hCG.
 29. The device of claim 25, wherein A) therelease medium comprises: i) a labeled conjugate comprising a detectablelabel and at least one binding member reactive with an epitope common toregular hCG and hCG-H; and ii) a capturable component comprising abinding member that exhibits a moderate to high affinity for hCG-H andis selectively or preferentially reactive with a second epitope ofhCG-H; and B) the capture medium comprises: i) a first capture sitehaving immobilized thereon a capture component having a binding affinityfor the capturable component; and ii) a second capture site comprisingan immobilized binding member that is reactive with a second epitope ofhCG.
 30. The device of claim 29, wherein the capturable componentcomprises a biotinylated capturable component and the capture componentcomprises streptavidin.
 31. The device of claim 25, wherein A) therelease medium comprises: i) a labeled conjugate comprising a detectablelabel and at least one binding member reactive with an epitope common toregular hCG and hCG-H; and ii) a capturable component comprising abinding member that is selectively or preferentially reactive with asecond epitope of regular hCG; and B) the capture medium comprises: i) afirst capture site having immobilized thereon a capture component havinga binding affinity for the capturable component; and ii) a secondcapture site comprising an immobilized binding member that exhibits amoderate to high affinity for hCG-H and is selectively or preferentiallyreactive with a second epitope of hCG-H.
 32. The device of claim 31,wherein the capturable component comprises a biotinylated capturablecomponent and the capture component comprises streptavidin.
 33. A devicefor selectively detecting hyperglycosylated human chorionic gonadotropin(hCG-H) in a liquid sample, the device comprising: A) a common fluidpath for receiving and distributing the liquid sample; B) at least onerelease medium in fluid communication with the common fluid path, the atleast one release medium being formed of a first material and comprisinga detectable label; C) a first capture medium in fluid communicationwith the at least one release medium and formed of a second material,the first capture medium comprising a capture site that directly orindirectly selectively or preferentially binds hCG-H; and D) a secondcapture medium in fluid communication with the at least one releasemedium and formed of the second material, the second capture mediumcomprising a capture site that directly or indirectly binds regular hCG.34. The device of claim 33, wherein: A) the at least one release mediumcomprises: i) a first release medium comprising a labeled conjugatecomprising a detectable label and a binding member that is reactive witha first epitope of hCG-H; and ii) a second release medium comprising alabeled conjugate comprising a detectable label and a binding memberthat is reactive with a first epitope of regular hCG; B) the firstcapture medium being in fluid communication with the first releasemedium, the first capture medium comprising a capture site comprising abinding member that exhibits a moderate to high affinity for hCG-H andis selectively or preferentially reactive with a second epitope ofhCG-H; and C) the second capture medium being in fluid communicationwith the second release medium, the second capture medium comprising acapture site comprising a binding member that is selectively orpreferentially reactive with a second epitope of regular hCG.
 35. Thedevice of claim 33, wherein: A) the at least one release mediumcomprises: i) a first release medium comprising a labeled conjugatecomprising a detectable label and a binding member that is reactive witha first epitope of hCG-H, and further comprising a capturable componentcomprising a binding member that exhibits a moderate to high affinityfor hCG-H and is selectively or preferentially reactive with a secondepitope of hCG-H; and ii) a second release medium comprising a labeledconjugate comprising a detectable label and a binding member that isreactive with a first epitope of regular hCG; B) the first capturemedium being in fluid communication with the first release medium, thefirst capture medium comprising a capture site having immobilizedthereon a capture component having a binding affinity for the capturablecomponent; and C) the second capture medium being in fluid communicationwith the second release medium, the second capture medium comprising acapture site comprising a binding member that is reactive with a secondepitope of regular hCG.
 36. The device of claim 35, wherein thecapturable component comprises a biotinylated capturable component andthe capture component comprises streptavidin.
 37. The device of claim33, comprising a single release medium in fluid communication with boththe first capture medium and the second capture medium, the releasemedium comprising a labeled conjugate comprising a detectable label anda binding member that is reactive with a first epitope of hCG-H and afirst epitope of regular hCG.
 38. The device of claim 33, wherein thefirst capture medium comprises a capture site comprising a bindingmember that exhibits a moderate to high affinity for hCG-H and isselectively or preferentially reactive with a second epitope of hCG-H;and the second capture medium comprises a capture site comprising abinding member that is reactive with a second epitope of regular hCG.39. A method of evaluating the viability of a pregnancy, the methodcomprising: A) providing a test device comprising the device of claim 1;B) applying to the device a liquid sample potentially including one orboth of regular hCG and hCG-H; and C) detecting the presence or lackthereof of hCG-H in the liquid sample, wherein the detected presence ofhCG-H indicates a high probability of successful implantation andtherefore a viable pregnancy.
 40. A method of evaluating the viabilityof a pregnancy, the method comprising: A) providing a test devicecomprising the device of claim 2; B) applying to the device a liquidsample potentially including one or both of regular hCG and hCG-H; andC) detecting the presence or lack thereof of hCG-H in the liquid sample,wherein the detected presence of hCG-H indicates a high probability ofsuccessful implantation and therefore a viable pregnancy.
 41. A methodof evaluating the viability of a pregnancy, the method comprising: A)providing a test device comprising the device of claim 11; B) applyingto the device a liquid sample potentially including one or both ofregular hCG and hCG-H; and C) detecting the presence or lack thereof ofhCG-H in the liquid sample, wherein the detected presence of hCG-Hindicates a high probability of successful implantation and therefore aviable pregnancy.
 42. A method of evaluating the viability of apregnancy, the method comprising: A) providing a test device comprisingthe device of claim 22; B) applying to the device a liquid samplepotentially including one or both of regular hCG and hCG-H; and C)detecting the presence or lack thereof of hCG-H in the liquid sample,wherein the detected presence of hCG-H indicates a high probability ofsuccessful implantation and therefore a viable pregnancy.
 43. A methodof evaluating the viability of a pregnancy, the method comprising: A)providing a test device comprising the device of claim 25; B) applyingto the device a liquid sample potentially including one or both ofregular hCG and hCG-H; and C) detecting the presence or lack thereof ofhCG-H in the liquid sample, wherein the detected presence of hCG-Hindicates a high probability of successful implantation and therefore aviable pregnancy.
 44. A method of evaluating the viability of apregnancy, the method comprising: A) providing a test device comprisingthe device of claim 33; B) applying to the device a liquid samplepotentially including one or both of regular hCG and hCG-H; and C)detecting the presence or lack thereof of hCG-H in the liquid sample,wherein the detected presence of hCG-H indicates a high probability ofsuccessful implantation and therefore a viable pregnancy.